Rapid and accurate Bayesian diagnosis of heparin-induced thrombocytopenia.

Prompt diagnostic evaluation of suspected heparin-induced thrombocytopenia (HIT) is critical for guiding initial patient management. We assessed the performance of 3 immunoassays detecting anti-platelet factor 4 (PF4)/heparin antibodies, derived a diagnostic algorithm with a short analytical turnaround time (TAT), and prospectively validated the algorithm. Plasma samples were analyzed by Zymutest-HIA-IgG, HemosIL-AcuStar-HIT-IgG, and ID-H/PF4-PaGIA in retrospective (n = 221) and prospective (n = 305) derivation cohorts. We calculated likelihood ratios of result intervals and cutoff values with 100% negative (NPV) and positive (PPV) predictive values for a positive gold standard functional assay (heparin-induced platelet activation [HIPA]). A diagnostic algorithm was established based on the Bayesian combination of pretest probability and likelihood ratios of first- and second-line immunoassays. Cutoffs with 100% PPV for positive HIPA were >3.0 U/mL (HemosIL-AcuStar-HIT-IgG) and titer ≥16 (ID-H/PF4-PaGIA); cutoffs with 100% NPV were <0.13 U/mL and ≤1, respectively. During the prospective validation of the derived algorithm (n = 687), HemosIL-AcuStar-HIT-IgG was used as unique testing in 566 (82.4%) of 687 cases (analytical TAT, 30 minutes). In 121 (17.6%) of 687 unresolved cases, ID-H/PF4-PaGIA was used as second-line testing (additional TAT, 30 minutes). The algorithm accurately predicted HIT in 51 (7.4%) of 687 patients and excluded it in 604 (87.9%) of 687 patients, leaving only 20 (2.9%) cases unresolved. We also identified 12 (1.7%) of 687 positive predictions not confirmed by HIPA: 10 patients with probable HIT despite negative HIPA and 2 possible false-positive algorithm predictions. The combination of pretest probability with first- and second-line immunoassays for anti-PF4/heparin antibodies is accurate for ruling in or out HIT in ≥95% of cases within 60 minutes. This diagnostic approach improves initial management of patients with suspected HIT.

Generation of procoagulant collagen- and thrombin-activated platelets in platelet concentrates derived from buffy coat: the role of processing, pathogen inactivation, and storage.

BACKGROUND: Collagen- and thrombin-activated (COAT) platelets (PLTs), generated by dual-agonist stimulation with collagen and thrombin (THR), enhance THR generation at the site of vessel wall injury. There is evidence that higher amounts of procoagulant COAT PLTs are associated with stroke, while a decreased ability to generate them is associated with bleeding diathesis. Our aim was to study PLT functions, particularly the ability to generate COAT PLTs, in PLT concentrates (PCs) from buffy coat. Thus, we investigated the effect of processing, pathogen inactivation treatment (amotosalen-UVA), and PC storage. STUDY DESIGN AND METHODS: Two PCs from five donors each were pooled and split in two bags; one of them was pathogen inactivated and the other one was left untreated (n = 5). Flow cytometric analyses were performed immediately after PC preparation (Day 1) and thereafter on Days 2, 5, 7, and 9 in treated and untreated PCs to measure the reactivity of PLTs (CD62P and PAC-1), the content and secretion of dense granule after stimulation with different agonists, and the percentage of COAT PLTs after dual stimulation with convulxin (agonist of the collagen receptor GPVI) and THR. RESULTS: Preparation of PCs resulted in a significant decrease of COAT PLTs and in an impaired response to adenosine 5'-diphosphate sodium (ADP). Storage further decreased ADP response. Minor differences were observed between untreated or amotosalen-UVA-treated PCs. CONCLUSION: Preparation of PCs from buffy coats decreased the ability to generate COAT PLTs and impaired PLT response to ADP.

The Role of Plasma Transfusion in Massive Bleeding: Protecting the Endothelial Glycocalyx?

Massive hemorrhage is a leading cause of death worldwide. During the last decade several retrospective and some prospective clinical studies have suggested a beneficial effect of early plasma-based resuscitation on survival in trauma patients. The underlying mechanisms are unknown but appear to involve the ability of plasma to preserve the endothelial glycocalyx. In this mini-review, we summarize current knowledge on glycocalyx structure and function, and present data describing the impact of hemorrhagic shock and resuscitation fluids on glycocalyx. Animal studies show that hemorrhagic shock leads to glycocalyx shedding, endothelial inflammatory changes, and vascular hyper-permeability. In these animal models, plasma administration preserves glycocalyx integrity and functions better than resuscitation with crystalloids or colloids. In addition, we briefly present data on the possible plasma components responsible for these effects. The endothelial glycocalyx is increasingly recognized as a critical component for the physiological vasculo-endothelial function, which is destroyed in hemorrhagic shock. Interventions for preserving an intact glycocalyx shall improve survival of trauma patients.

Hepcidin as a new biomarker for detecting autologous blood transfusion.

Autologous blood transfusion (ABT) is an efficient way to increase sport performance. It is also the most challenging doping method to detect. At present, individual follow-up of haematological variables via the athlete biological passport (ABP) is used to detect it. Quantification of a novel hepatic peptide called hepcidin may be a new alternative to detect ABT. In this prospective clinical trial, healthy subjects received a saline injection for the control phase, after which they donated blood that was stored and then transfused 36 days later. The impact of ABT on hepcidin as well as haematological parameters, iron metabolism, and inflammation markers was investigated. Blood transfusion had a particularly marked effect on hepcidin concentrations compared to the other biomarkers, which included haematological variables. Hepcidin concentrations increased significantly: 12 hr and 1 day after blood reinfusion, these concentrations rose by seven- and fourfold, respectively. No significant change was observed in the control phase. Hepcidin quantification is a cost-effective strategy that could be used in an "ironomics" strategy to improve the detection of ABT.

Urinary di-(2-ethylhexyl) phthalate metabolites for detecting transfusion of autologous blood stored in plasticizer-free bags.

BACKGROUND: Autologous blood transfusion (ABT) efficiently increases sport performance and is the most challenging doping method to detect. Current methods for detecting this practice center on the plasticizer di(2-ethlyhexyl) phthalate (DEHP), which enters the stored blood from blood bags. Quantification of this plasticizer and its metabolites in urine can detect the transfusion of autologous blood stored in these bags. However, DEHP-free blood bags are available on the market, including n-butyryl-tri-(n-hexyl)-citrate (BTHC) blood bags. Athletes may shift to using such bags to avoid the detection of urinary DEHP metabolites. STUDY DESIGN AND METHODS: A clinical randomized double-blinded two-phase study was conducted of healthy male volunteers who underwent ABT using DEHP-containing or BTHC blood bags. All subjects received a saline injection for the control phase and a blood donation followed by ABT 36 days later. Kinetic excretion of five urinary DEHP metabolites was quantified with liquid chromatography coupled with tandem mass spectrometry. RESULTS: Surprisingly, considerable levels of urinary DEHP metabolites were observed up to 1 day after blood transfusion with BTHC blood bags. The long-term metabolites mono-(2-ethyl-5-carboxypentyl) phthalate and mono-(2-carboxymethylhexyl) phthalate were the most sensitive biomarkers to detect ABT with BTHC blood bags. Levels of DEHP were high in BTHC bags (6.6%), the tubing in the transfusion kit (25.2%), and the white blood cell filter (22.3%). CONCLUSIONS: The BTHC bag contained DEHP, despite being labeled DEHP-free. Urinary DEHP metabolite measurement is a cost-effective way to detect ABT in the antidoping field even when BTHC bags are used for blood storage.

High-dose therapy and autologous hematopoietic stem cell transplant in T-cell lymphoma: a single center experience.

We report here the long-term outcome of autologous stem cell transplant in peripheral T-cell lymphoma (PTCL). Forty-three consecutive patients with PTCL diagnosed between 2000 and 2011 were treated with high-dose chemotherapy (HDCT) and autologous stem cell transplant (ASCT) in our center. Diagnoses included PTCL-not otherwise specified (n = 19), anaplastic large cell lymphoma (n = 11), angioimmunoblastic T-cell lymphoma (n = 5), enteropathy-associated T-cell lymphoma (n = 5) and other rare subtypes (n = 3). Thirty-six patients with a median age of 50 years (range 22-65) were transplanted in first response and seven after relapse. After a median follow-up of 63 months, estimated overall survival at 12 years was 40%, progression-free survival at 12 years was 34% and event-free survival at 12 years was 30%. On univariate analysis, age less than 50 years and no B symptoms at diagnosis were significantly associated with prolonged overall and progression-free-survival. HDCT/ASCT for peripheral T-cell lymphoma can lead to long-term survival for patients responding to induction chemotherapy.

Oxidation of proteins: Basic principles and perspectives for blood proteomics.

Protein oxidation mechanisms result in a wide array of modifications, from backbone cleavage or protein crosslinking to more subtle modifications such as side chain oxidations. Protein oxidation occurs as part of normal regulatory processes, as a defence mechanism against oxidative stress, or as a deleterious processes when antioxidant defences are overcome. Because blood is continually exposed to reactive oxygen and nitrogen species, blood proteomics should inherently adopt redox proteomic strategies. In this review, we recall the biochemical basis of protein oxidation, review the proteomic methodologies applied to analyse redox modifications, and highlight some physiological and in vitro responses to oxidative stress of various blood components.

Plasma/serum proteomics: pre-analytical issues.

High-throughput proteomics technologies tend to provide highly sensitive information about living tissues and biological fluids. Analytes are characterized by intrinsic and extrinsic properties, the latter depending on each phase of their preparation, sometimes adding artifacts with crucial repercussions in result reliability and interpretation. This review aims to address some issues that can be encountered when handling plasma and serum in experimental and clinical proteomic settings.

Proteomic analyses of amniotic fluid: potential applications in health and diseases.

Amniotic fluid (AF) is a potential source of biomarkers for many disorders which may occur during pregnancy. The purpose of this study was to evaluate the place of two-dimensional gel electrophoresis (2-DE) technologies to compare AF in both normal and pathological situations. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE; Ettan DIGE) as well as two-dimensional gel electrophoresis and silver staining followed by image analysis were used. Differentially expressed proteins were identified by mass spectrometry. This approach was used to study electrophoregrams of normal AF obtained at 17 weeks of gestation and at term, as well as AF from fetuses presenting with congenital diaphragmatic hernia. Finally, the potential of two-dimensional electrophoresis was assessed by studying the protein profile of plasma containing AF proteins in a model of premature rupture of the membranes (PROM). Our results clearly show that two-dimensional electrophoresis technologies still have place for analyzing biological fluids such as AF.

Proteomics and transfusion medicine: future perspectives.

Limited number of important discoveries have greatly contributed to the progresses achieved in the blood transfusion; ABO histo-blood groups, citrate as anticoagulant, fractionation of plasma proteins, plastic bags and apheresis machines. Three major types of blood products are transfused to patients: red cell concentrates, platelet concentrates and fresh frozen plasma. Several parameters of these products change during storage process and they have been well studied over the years. However, several aspects have completely been ignored; in particular those related to peptide and protein changes. This review presents what has been done using proteomic tools and the potentials of proteomics for transfusion medicine.

Clinical proteomics: study of a cryogel.

Cryoproteins are proteins precipitating at low temperature. Usually, the precipitate contains immunoglobulins (Igs), and are therefore called cryoglobulins. Very rarely, Igs do not precipitate, but, upon cooling, form a gel. Here, we report a case of cryogel observed in a patient presenting with Waldenström's disease. Using proteomic tools, a monoclonal IgM was identified as being the cause of the gel formation. Furthermore, addition of H(2)O before incubation at 4 degrees C demonstrated that the monoclonal IgM was precipitable as a type I cryoglobulin (hypocryoglobulin).