International Council for Standardization in Haematology Field Study Evaluating Optimal Interpretation Methods for Activated Partial Thromboplastin Time and Prothrombin Time Mixing Studies.

CONTEXT.­: The prothrombin time (PT) and activated partial thromboplastin time (APTT) are screening tests used to detect congenital or acquired bleeding disorders. An unexpected PT and/or APTT prolongation is often evaluated using a mixing test with normal plasma. Failure to correct ("noncorrection") prolongation upon mixing is attributed to an inhibitor, whereas "correction" points to factor deficiency(ies). OBJECTIVE.­: To define an optimal method for determining correction or noncorrection of plasma mixing tests through an international, multisite study that used multiple PT and APTT reagents and well-characterized plasma samples. DESIGN.­: Each testing site was provided 22 abnormal and 25 normal donor plasma samples, and mixing studies were performed using local PT and APTT reagents. Mixing study results were evaluated using 11 different calculation methods to assess the optimal method based on the expected interpretation for factor deficiencies (correction) and noncorrection (inhibitor effect). Misprediction, which represents the failure of a mixing study interpretation method, was assessed. RESULTS.­: Percentage correction was the most suitable calculation method for interpreting PT mixing test results for nearly all reagents evaluated. Incubated PT mixing tests should not be performed. For APTT mixing tests, percentage correction should be performed, and if the result indicates a factor deficiency, this should be confirmed with the subtraction III calculation where the normal pooled plasma result (run concurrently) is subtracted from the mixing test result with correction indicated by a result of 0 or less. In general, other calculation methods evaluated that performed well in the identification of factor deficiency tended to have high misprediction rates for inhibitors and vice versa. CONCLUSIONS.­: No single method of mixing test result calculation was consistently successful in accurately distinguishing factor deficiencies from inhibitors, with between-reagent and between-site variability also identified.

Association of Fibrinolytic Potential and Risk of Mortality in Cancer Patients.

Cancer is a leading cause of death, and the fibrinolytic system shows cooperative effects that facilitate the growth of tumors and the appearance of metastases. This prospective study aimed to evaluate the fibrinolytic potential in cancer patients and its association with mortality outcomes using the fluorometric method of simultaneous thrombin and plasmin generation. The study included 323 cancer patients and 148 healthy individuals. During the 12-month follow-up, 68 patients died. Compared to the control group, cancer patients showed alterations in thrombin production consistent with a hypercoagulability profile, and an increase in plasmin generation. Mortality risk was associated with two parameters of thrombin in both univariate and multivariable analysis: maximum amplitude (Wald 11.78, p < 0.001) and area under the curve (Wald 8.0, p < 0.005), while such associations were not observed for plasmin. In conclusion, this was the first study able to demonstrate the simultaneous evaluation of thrombin and plasmin generation in newly diagnosed untreated cancer patients. Patients with cancer have been observed to exhibit a hypercoagulable profile. During the study, two parameters linked to thrombin generation, MA and AUC, were identified and found to have a potential association with mortality risk. However, no associations were found with parameters related to plasmin generation.

International Council for Standardization in Haematology recommendations for laboratory measurement of factor VIII and FIX type I inhibitors.

This guidance document has been prepared on behalf of the International Council for Standardisation in Hematology. The aim of the document is to provide guidance and recommendations on the measurement of factor VIII (FVIII) and factor IX (FIX) inhibitors. After an introduction on the clinical background and relevance of factor VIII and factor IX inhibitor testing, the following aspects of laboratory testing are included: screening for inhibitors, assay principle, sample requirements, testing requirements and interpretation, quality assurance, interferences and recent developments. This guidance document focusses on recommendations for a standardised procedure for the laboratory measurement of FVIII and FIX type I inhibitors. The recommendations are based on published data in peer-reviewed literature and expert opinion.

Activated Partial Thromboplastin Time and Prothrombin Time Mixing Studies: Current State of the Art.

Mixing studies have long been in the clinical laboratory armamentarium for investigating unexpected, prolonged activated partial thromboplastin time (aPTT) or prothrombin time (PT). The purpose of the mixing study is to identify whether the aPTT/PT prolongation is secondary to a factor deficiency versus an inhibitor, which would present as a "corrected" and "noncorrected" mixing study, respectively. The differentiation between a factor deficiency and inhibitor may likely further direct clinical decisions, including additional diagnostic testing or factor replacement therapy. While aPTT/PT mixing studies are simple tests to perform, there is a lack of standardization for both the testing protocol and the interpretation of what is considered to be a corrected or noncorrected mixing study result. This review will describe the common indications for the mixing test, preanalytic variables that may affect mixing study performance, and describe several methods for interpreting the results of aPTT and PT mixing tests.

Advantages of external quality assessment-EQA programs.

INTRODUCTION: The first external quality assessment (EQA) in Thrombosis and Haemostasis was elaborated over 20 years ago, and since then, several national and international EQA institutions have been established. AIM: Display the benefits of EQA programs. METHODS: The spectrum of EQA action was evaluated ranges from improving the performance of the local laboratory to highlighting inadequate diagnostic tests that need to be replaced by new technologies. RESULTS: The first result approach is related to a national management of quality in laboratories. In recent years, Brazil has invested in an EQA program to aid public policy in the laboratory area. During this period, a group of haemostasis laboratory specialists were invited to manage the results and help the Ministry of Health with applying these results as a strategy to improve laboratories. Thus, in collaboration with NEQAS-BC, the University of Campinas - UNICAMP, established a Brazilian EQA program for Blood Coagulation. The second result approach is related to FVIII inhibitor assay performance evaluation, which is another type of EQA program benefit. Despite the assay being considered the gold standard to measure neutralised immunoglobulins for FVIII since 1975, over 40 years ago, the test still has a high coefficient of variation. Results from NEQAS-BC and WFH IEQAS program demonstrate the inter-laboratory variation across the United Kingdom over the last years and among emergent countries. CONCLUSION: The EQA programs have an important educational role in helping countries manage their public policy and in the international inquiry regarding the necessity of new technologies in haemostasis.

The role of platelet-rich plasma in the mild and severe stages of atherosclerotic disease in mice.

Background: Platelet-rich plasma (PRP) has a high concentration of growth factors (GFs), which present a therapeutic wound healing effect. Despite having been correlated with an immunomodulatory function, the administration of PRP has not yet been investigated in atherosclerosis models. Aim: Evaluate the effect of lyophilized PRP on atherosclerosis in mice models through serum analysis. Methods: Animals received a high-fat diet for disease induction and a weekly PRP retro-orbital application. Effectiveness was evaluated by measuring inflammatory markers in plasma following the treatment of mice with either PRP or saline solution. Results: PRP was well characterized for platelet and GF concentrations; the atherosclerotic profile was established. Cytokine concentrations were altered after PRP applications. Conclusion: PRP could modulate the inflammatory pattern in the early stages of atherosclerosis.

Platelet-rich plasma (PRP) contains growth factors, which stimulate normal wound healing. This product seems to be a good modulator of white blood cells and has not been investigated in atherosclerosis. This study aimed to evaluate PRP in atherosclerosis using mice models. The PRP was produced from animals and preserved using the lyophilization technique; the product was then applied weekly in the vein. For atherosclerosis induction, genetically modified animals were fed a high-fat diet. The effectiveness was evaluated by measuring plasma inflammatory markers after treatment, and PRP seemed to alter cell-signaling molecules (cytokines). This study concluded that PRP was capable of modulating the inflammatory pattern during the early stages of atherosclerosis.

Neutrophil activation and circulating neutrophil extracellular traps are increased in venous thromboembolism patients for at least one year after the clinical event.

Neutrophil activation and neutrophil extracellular traps (NETs) have been associated with the pathogenesis of venous thromboembolism (VTE). Considering VTE-associated chronic sequelae, which suggest that some pathological mechanisms remain after the acute episode, we investigated whether neutrophil activation is increased in patients with a prior VTE at least one year before this investigation. Thirty-seven patients with prior VTE and 37 individuals with no history of VTE were included. Neutrophil activity was evaluated by the expression of the adhesive molecule activation-specific epitopes LFA-1 (CD11a) and MAC-1 (CD11b), chemotaxis, reactive oxygen species (ROS) and by MPO-DNA complexes as markers of NETs. The adhesive molecules sICAM-1 and sVCAM-1, involved in the cross talk between neutrophil and endothelial cells, were also evaluated. Patient neutrophils presented increased CD11a expression before and after TNF-α stimulus, whereas increased CD11b expression was observed only after TNF-α stimulus, as compared to controls. Neutrophil chemotaxis on both, basal state and after IL-8 stimulus, on circulating levels of sICAM-1 and sVCAM-1, and on MPO-DNA complexes were also increased in VTE patients. ROS release was similar between patients and controls. This is, to our knowledge, the first study to investigate neutrophil inflammatory activity in VTE patients a long period after an acute event (approximately 2 years). The results showed altered neutrophil activation patterns in these patients. While activated neutrophils can cause endothelial activation and injury, the activated endothelium can induce the release of NETs with consequent endothelial cytotoxicity, creating a vicious cycle of activation between neutrophils and endothelium that can lead to thrombosis. VTE patients (approximately 2 years after the clinical event) present an altered neutrophil activation state evidenced by increased activity of the LFA-1 and Mac-1 adhesive molecules, as well as increased chemotaxis and circulating levels of NETs remnants. Circulating levels of ICAM-1 and VCAM-1, which are endothelial adhesive molecules, are also increased in VTE patients, suggesting not only an exacerbated endothelial activation and dysfunction, but also an interaction of the neutrophil adhesive molecules with their endothelial ligands, favoring the migration process of neutrophil.

Serum Metabolic Profiles Based on Nuclear Magnetic Resonance Spectroscopy among Patients with Deep Vein Thrombosis and Healthy Controls.

Deep venous thrombosis (DVT) is associated with significant morbidity and mortality. Studies on changes in the level of metabolites could have the potential to reveal biomarkers that can assist in the early detection, diagnosis, monitoring of DVT progression, response to treatment, or recurrence of DVT. In this scenario, the metabolomic analysis can provide a better understanding of the biochemical dysregulations of thrombosis. Using an untargeted metabolomic approach through magnetic resonance spectroscopy and multi- and univariate statistical analysis, we compared 40 patients with previous venous thrombosis and 40 healthy individuals, and we showed important serum differences between patients and controls, especially in the spectral regions that correspond to glucose, lipids, unsaturated lipids, and glycoprotein A. Considering the groups depending on risk factors and the local of the previous episode (lower limbs or cerebral system), we also noticed differences in metabolites linked to lipids and lactate. Comparative analyses pointed to altered ratios of glucose/lactate and branched-chain amino acids (BCAAs)/alanine, which might be associated with the fingerprints of thrombosis. Although samples for metabolomic analysis were collected months after the acute episode, these results highlighted that, alterations can still remain and may contribute to a better understanding of the complications of the disease.

Characterization of autologous platelet rich plasma (PRP) and its biological effects in patients with Behçet's Disease.

INTRODUCTION: Behçet's disease (BD) is an immune-mediated chronic systemic vasculitis, characterized by clinical manifestations that include: mucocutaneous ulcers, ocular involvement, immunological alterations, vascular and neurological implications. The available treatments present limitations such as high cost and side effects, and the search for a low-cost biological treatment with immunomodulatory potential becomes of great value. Platelet rich plasma (PRP) has some characteristics that indicate a possible use as an immunomodulator due to the wide range of secreted cytokines, especially through the participation of TGF-ß1 in the differentiation of T regulatory cells (Treg). This study aimed to characterize the PRP poor in leukocytes (P-PRP) of patients with BD and active ulcers and to evaluate its effects as an immunomodulator through a subcutaneous application. METHODS: We selected patients with a diagnosis of BD, with a low dose of prednisone and with no central nervous system or ocular involvement. Platelet and leukocyte count and quantification of 17 cytokines were evaluated in P-PRP. The effects of P-PRP were evaluated by cell frequency of TCD4 +, TCD8 +, Treg, natural killer (NK), and activated NK, as well as by the cytokine profile in patient's plasma, and the clinical manifestations through score and questionnaire. Also, it was evaluated the number and timing of oral ulcer closure. PRP was used as an adjuvant, with 9 applications of 3 mL, over 6 months, with a follow-up of one year. RESULTS: The results using PRP showed adequate values and no significant inter-and intra-individual variations. The systemic evaluations during the use of PRP showed significant alterations, characterized by the increase in Treg cell frequency (p = 0.0416) and a decrease in activated NK cells (p = 0.0010). However, no clinical correlation was observed through score analysis. The most relevant clinical data was the decrease in the closing time of ulcers throughout the application period. CONCLUSION: In a pilot study with BD patients, P-PRP promoted an anti-inflammatory profile characterized by increased Treg cells and decreased activated NK cells and alterations in cytokines. A clinical improvement was observed with a decrease in the number and time of closure of oral ulcers.

A case series of platelet rich plasma in chronic venous ulcers.

INTRODUCTION: Venous ulcers are the most common type of leg wounds (80%) and the main cause is chronic venous insufficiency. Autologous platelet-rich plasma (PRP) is a potential wound healing treatment due to its great variety of growth factors. The aim of this study was to describe in a case series the results of poor-leukocyte PRP (P-PRP) or saline for the treatment of chronic non-healing ulcers of the lower extremity. METHODS: Eight patients were treated according to the topical therapy: saline solution or P-PRP gel. All patients used double compression stocks and were assisted by a vascular practitioner for up to 12 months or until wound healing. The treatment was performed weekly with cleaning of the affected area, macroscopic evaluation (area measurement and photos) and P-PRP or saline application, and closure with Tegaderm®. Trial Registration: Retrospectively approved by Brazilian Clinical Trials, register number RBR-7zhgb3 (http://www.ensaiosclinicos.gov.br/rg/RBR-7zhgb3/). RESULTS: All patients showed signs of wound healing with a reduction in wound size and ulcer numbers, but more evident with P-PRP application. CONCLUSIONS: The results suggested that P-PRP presented a better result when compared to saline solution in the healing process of long clinical course chronic venous ulcers, when associated to compressive stocks and topical care.

The effect of lyophilized platelet rich-plasma on skin aging: a non-randomized, controlled, pilot trial.

Platelet-rich plasma (PRP) showed positive results in the improvement of skin aging. Lyophilized PRP can be interesting in clinical practice due to the facility to obtain many samples in a single blood collection and can be used in multiple injections. To evaluate the effect of lyophilized PRP in the treatment of skin aging, through a Phase II pilot study. Nineteen women (54 years ± 7 years) with Glogau photoaging II and III types were select for this non-randomized, split-face controlled study. They received monthly intradermal injections of lyophilized PRP and saline solution (as control) into the facial skin, during a period of 2 months. The evaluation was performed by imaging method, histological techniques, and multiphoton microscopy. Although lyophilized PRP presented 10 times the platelet baseline value (P < .0001) and growth factors in adequate levels, only saline solution showed an increase of dermis thickness (p = .0009). Collagen pre and post-application remained the same for both types of treatments. The use of lyophilized PRP by mesotherapy showed no improvement on skin aging. TRIAL REGISTRATION APPROVAL: RBR-3n9wxw, UTN U1111-1226-6093-retrospectively registered.

From Hemophilia to Deep Venous Thrombosis Patient Samples: How to Perform an Easy Coagulometer Validation Process According to Available Guidelines.

Validation protocols for the evaluation of coagulometers are needed to help professionals select the most suitable system for their regular laboratory routines. The objective of this study was to show how high standard protocols for the coagulometer validation process can fit into the daily laboratory routine. For this study, 45 healthy individuals and 112 patient samples were analyzed. From the patient samples, 51 were investigated for deep venous thrombosis, 27 for coagulopathy, 19 for antivitamin K therapy, and 15 for hemophilia. For the assessment, the performance of the 3 coagulometers and 1 point-of-care device was considered. One of the coagulometers was a new acquisition evaluated for precision, linearity, throughput, and carryover in the first moment, and the new coagulometer was then compared with the other well-established equipment in the laboratory. In normal plasma, coefficient of variation was ≤1.8% for total precision in screening tests and ≤3.5% for within-run precision in specific assays. For prothrombin time/international normalized ratio, no significant difference was found when comparing methods. Our study showed how to compare the capacity of a reagent in order to discriminate patients with severe hemophilia from patients with moderated hemophilia, and the κ coefficient agreement was 0.669 (95% confidence interval: 0.3-1.0; P < .001). d-dimer evaluated in patients with deep venous thrombosis and controls showed a 20% discrepancy between the methods. In our experience across Latin America, the number of laboratories that has performed this process is limited. In this study, we demonstrated how to adapt the validation process for the hemostasis laboratory routine to help the professional chose the best and more suitable option.

Increased inflammation and endothelial markers in patients with late severe post-thrombotic syndrome.

INTRODUCTION: Post-thrombotic syndrome (PTS) is a limiting long-term complication present in 20-50% of patients with deep venous thrombosis (DVT) of the lower limbs. A panel of biomarkers with potential relevance to enhance knowledge on the pathophysiology of PTS was investigated. METHODS: This case-control study included 93 patients with DVT in the lower limbs, 31 with severe PTS (cases) and 62 with mild/no PTS (controls), over 24 months after an acute episode. Thirty-one healthy individuals (HI) with no history of DVT were included as a reference to the population. FVIII activity, D-dimer, inflammatory cytokines, endothelial dysfunction markers, matrix metalloproteinases, and their inhibitors, tissue remodeling and growth factor levels were evaluated. The classification of PTS was, by the Villalta scale. RESULTS: Patients with severe PTS showed elevated levels of CRP, sICAM-1, sE-selectin, and decreased MMP-9 and MCP-1 levels when compared to patients with mild/no PTS. Moreover, DVT patients presented higher levels of FVIII and D-dimer when compared to HI. CONCLUSIONS: DVT patients present an inflammatory status, endothelial dysfunction and altered proteolysis MMPs activity, even a long time after the acute thrombotic episode, which is more significant in severe PTS. These results suggest a possible role of these mediators in the maintenance and worsening of PTS severity.

Low prevalence of Post-thrombotic syndrome in patients treated with rivaroxaban.

Post-thrombotic syndrome (PTS) is a complication of deep vein thrombosis (DVT). Residual vein thrombus (RVT) on Doppler Ultrasound can be associated with PTS. Limited data are available on the effect of direct oral anticoagulants (DOACs) on the long-term outcome of PTS. This study aimed to compare the prevalence of PTS and RVT, in patients with previous DVT treated with rivaroxaban or enoxaparin/warfarin. A total of 129 patients with previous proximal lower limb DVT and treated with rivaroxaban (n = 71) or enoxaparin/warfarin (n = 58) for at least 3 months were included. The Villalta scale for PTS was performed after treatment. The median duration of the DVT symptoms before anticoagulation was 7 days for both groups. The rate of PTS was 50.7% in the patients treated with rivaroxaban and 69% in the enoxaparin/warfarin group. Enoxaparin/warfarin showed an increased prevalence of PTS (P = .018). An analysis in 3 different models showed that the relative risk of PTS decreased by 76% with rivaroxaban use when compared with enoxaparin/warfarin treatment. In addition, 93 of the 129 patients were evaluated regarding the presence of RVT, of which, 11 (24.4%) and 31 (64.6%) presented with RVT for rivaroxaban and enoxaparin/warfarin, respectively (P < .0001). The RVT analysis excluded the possibility of RVT as a mediator of the association between type of treatment and PTS when comparing rivaroxaban with enoxaparin/warfarin (odds ratio (OR) = 0.14; 95% confidence interval (CI): 0.1-1.0, P = .051) with rivaroxaban compared with enoxaparin/warfarin. Rivaroxaban treatment was associated with a lower risk of PTS when compared to enoxaparin/warfarin; RVT however, was not a mediator in the association between PTS and type of treatment.

Prolonged APTT of unknown etiology: A systematic evaluation of causes and laboratory resource use in an outpatient hemostasis academic unit.

BACKGROUND: A prolonged activated partial thromboplastin time (APTT) of unknown cause is one of the most frequent reasons why outpatients are referred for hemostasis consultation. Nevertheless, very few data are available on the relative contribution of individual causes of this common clinical scenario. Here, we present a systematic evaluation of all causes of APTT prolongation in a consecutive population of outpatients referred for specialized hemostasis consultation during a 14-year period. METHODS: All cases referred to an academic specialized hemostasis outpatient unit due to APTT prolongation of unknown etiology whose prolonged APTT was confirmed in the first visit were included in the study. Data were obtained from the electronic medical records. RESULTS: Among 187 consecutive patients, the most frequent causes were antiphospholipid antibodies in 22.6%, contact pathway factor deficiencies in 17.4%, other coagulation factor deficiencies in 11.6%, and vitamin K deficiency/liver disease in 11.6%. A definite cause was not identified in 22.1% of patients. Presence of antiphospholipid antibodies, and absence of bleeding symptoms were both associated with significantly longer APTT values compared to other categories/clinical scenarios. The investigation of each case required a mean of 18.2 additional tests per patient, with estimated costs ranging from US$191.60 to US$1055.60. CONCLUSIONS: Our results describe the main causes of APTT prolongation in outpatients, as well as estimates of resource use required to investigate this condition, thus providing evidence supporting the importance of measures to minimize the indiscriminate use of this assay.

Freeze-dried versus fresh platelet-rich plasma in acute wound healing of an animal model.

Aim: To compare freeze-dried and fresh platelet-rich plasma (PRP) preparations, in a pre-clinical study. Materials & methods: 30 Wistar male rats were used to compare and characterize human PRP which was applied at the perilesional area in an acute wound model, evaluated by macroscopical and histological analysis. Results: Despite the increased growth factor concentration after the freeze-drying process, no change in the healing kinetics was observed in vivo. Nevertheless, a significant increased number of myofibroblasts was demonstrated in comparison with the fresh PRP group. We also demonstrated a significant increased percentage of blood vessels in comparison with controls in both the superficial and deep epidermis. Conclusion: These results encourage randomized clinical trials to evaluate the effectiveness of freeze-dried PRP for skin ulcer treatment.

Treatment of male pattern alopecia with platelet-rich plasma: A double-blind controlled study with analysis of platelet number and growth factor levels.

BACKGROUND: Promising results with platelet-rich plasma (PRP) in androgenetic alopecia that could be associated with platelet number and growth factor levels were described. OBJECTIVE: Analyze the platelet countand growth factor levels in PRP and their correlation with hair growth parameters evaluated by using the TrichoScan (Tricholog GmbH, Freiburg, Germany). METHODS: A total of 26 patients were randomized to receive 4 subcutaneous injections of PRP or saline. Hair growth, hair density, and percentage of anagen hairs were evaluated by using the TrichoScan method before injection, 15 days after the last injection, and again 3 months after the last injection. Growth factors (platelet-derived growth factor, epidermal growth factor, and vascular endothelial growth factor) were measured by the Luminex method (Millipore, Bedford, MA). RESULTS: We demonstrated a significant increase in hair count (P = .0016), hair density (P = .012) and percentage of anagen hairs (P = .007) in the PRP group versus in the control group, without correlation with platelet counts or quantification of the growth factors in PRP. LIMITATIONS: Other growth factors that could be related to response to PRP were not evaluated. CONCLUSION: Our data favor the use of PRP as a therapeutic alternative in the treatment of androgenetic alopecia. The lack of association between platelet count, platelet-derived growth factor, epidermal growth factor, and vascular endothelial growth factor levels and clinical improvement suggest that other mechanisms could be involved in this response.