Acquired hemophilia A and delta storage pool deficiency in a patient with indolent non-Hodgkin lymphoma.

B-cell lymphoproliferative diseases may be associated with acquired hemostasis disorders, such as acquired hemophilia A (AHA) caused by autoantibodies that neutralize factor VIII activity, and δ-storage pool deficiency, an abnormality of platelet function due to defective dense granules and impaired secretion. We describe the case of a 67-year-old man in whom these two acquired bleeding disorders were concomitantly present as the first clinical manifestation of an indolent non-Hodgkin lymphoma. Immunosuppressive therapy with prednisone was initially started to eradicate anti-FVIII antibodies, subsequently boosted with cyclophosphamide and rituximab, these medications being also chosen to treat the associated indolent lymphoma. Bleeding symptoms were first tackled with limited benefit by using rFVIIa and then rescued using recombinant porcine FVIII. After a 6 month's follow-up lymphoma and AHA were in remission and platelet function was improved. This case underlines the need of multiple and complex diagnostic and therapeutic approaches to rare acquired bleeding disorders associated with lymphoproliferative diseases.

Evaluation of platelet function in essential thrombocythemia under different analytical conditions.

Background. Studies of platelet aggregation (PA) in essential thrombocythemia (ET) reported contrasting results, likely due to differences in analytical conditions.Objective. We investigated platelet aggregation using different techniques and analytical conditions.Patients and Methods. PA was studied by light-transmission aggregometry (LTA) in platelet-rich plasma (PRP) and impedance aggregometry in PRP and whole blood (WB). ADP, collagen, thrombin receptor activating peptide (TRAP-14) and adrenaline were used as agonists. Since ET patients (n = 41) were on treatment with aspirin (100 mg/d), healthy controls (n = 29) were given aspirin (100 mg/d) for 5 days before testing: therefore, thromboxane A2-independent PA was tested in all subjects. Blood samples were collected in citrate (C) [low Ca2+] or lepirudin (L) [physiological Ca2+]; platelet count was adjusted to 250 x 109/L in a set of C-PRP (adjusted C-PRP) and left unmodified in the other samples.Results. Results of PA in 17 ET patients who were poor responders to aspirin (high serum thromboxane B2 levels) were not included in the analysis. With LTA, PA in ET was lower than in controls in adjusted C-PRP and normal in native C-PRP and L-PRP. With impedance aggregometry, PA in L-PRP and L-WB tended to be higher in ET than in controls. Platelet serotonin and ADP contents were reduced in ET. The percentages of circulating platelets expressing P-selectin and platelet-leukocyte hetero-aggregates were higher in ET.Conclusions. Analytical conditions dramatically affect in vitro PA of ET patients, which appears defective under the least physiological conditions and normal/supranormal under conditions that are closer to the physiological.

Blood Cell-Bound C4d as a Marker of Complement Activation in Patients With the Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is a chronic and disabling condition characterized by recurrent thrombosis and miscarriages mediated by antibodies against phospholipid-binding proteins (aPL), such as beta2glycoprotein I (ß2GPI). Complement is involved in APS animal models and complement deposits have been documented in placenta and thrombotic vessels despite normal serum levels. Analysis of circulating blood cells coated with C4d displays higher sensitivity than the conventional assays that measure soluble native complement components and their unstable activation products in systemic lupus erythematosus (SLE). As C4d-coated blood cell count has been reported to be more sensitive than serum levels of complement components and their activation products in systemic lupus erythematosus (SLE) patients, we decided to evaluate the percentage of C4d positive B lymphocytes (BC4d), erythrocytes (EC4d), and platelets (PC4d) in primary APS patients and asymptomatic aPL positive carriers as marker of complement activation in APS. We assessed by flow cytometry the percentages of BC4d, EC4d, and PC4d in primary APS (PAPS; n. 23), 8 asymptomatic aPL positive carriers, 11 APS-associated SLE (SAPS), 17 aPL positive SLE, 16 aPL negative SLE, 8 aPL negative patients with previous thrombosis, 11 immune thrombocytopenia (ITP) patients, and 26 healthy subjects. In addition, we used an in vitro model to evaluate the ability of a monoclonal anti-ß2GPI antibody (MBB2) to bind to normal resting or activated platelets and fix complement. EC4d and PC4d percentages were significantly higher in PAPS and aPL carriers as well as aPL positive SLE and SAPS than in aPL negative controls. The highest values were found in PAPS and in SAPS. The EC4d and PC4d percentages were significantly correlated with serum C3/C4 and anti-ß2GPI/anti-cardiolipin IgG. In vitro studies showed that MBB2 bound to activated platelets only and induced C4d deposition. The detection of the activation product C4d on circulating erythrocytes and platelets supports the role of complement activation in APS. Complement may represent a new therapeutic target for better treatment and prevention of disability of APS patients.

Aggregometry in the settings of thrombocytopenia, thrombocytosis and antiplatelet therapy.

A variety of laboratory tests have been developed, which can diagnose a number of both congenital and acquired disorders of platelet function. Many tests of platelet function measure the ability of platelets to adhere to each other, forming platelet aggregates, which represent the major constituents of hemostatic plugs and of arterial thrombi. Light transmission aggregometry (LTA) is still considered the gold standard of platelet aggregation tests, but other platelet aggregation-based tests are also available. Among them, the flow cytometry-based methods may be more convenient than LTA for the study of patients with very low or very high platelet counts. The use of platelet aggregation tests has also been advocated to monitor the treatment with antiplatelet agents (mostly the P2Y12 antagonist clopidogrel) of patients with thrombotic arterial occlusions, with the aim of improving their efficacy and safety. However, randomized clinical trials failed to show any advantage of this strategy; as a consequence, international guidelines now recommend against laboratory monitoring of antiplatelet therapy.

Switching from clopidogrel to prasugrel to protect early invasive treatment in acute coronary syndromes: Results of the switch over trial.

BACKGROUND: Clopidogrel is used to pretreat patients with non-ST elevation acute coronary syndromes (NSTE-ACS), but prasugrel provides better platelet inhibition with improved outcome. However, switching from clopidogrel at the time of percutaneous coronary intervention (PCI) remains incompletely defined. Our aim was to compare the pharmacodynamic (PD) effects of 3 prasugrel loading doses (LDs; G1:10mg, G2: 30mg, and G3: 60mg) before PCI. A fourth group, continuing clopidogrel, served as control (G4). METHODS: 100 clopidogrel-pretreated patients were enrolled and blood collected before PCI, 30min, 1, 2, 4, 6, 24 and 48h thereafter. Platelet inhibition was measured by vasodilator-stimulated phosphoprotein phosphorylation (VASP) and Verify-Now assays. The end-points (EP) was the difference of PD effect at 4h between G3 and G4 (primary EP) with hierarchic evaluation between G2 and G1 versus G4 (secondary EP). A mixed-design ANOVA statistic was used to compare the four group scores over time. RESULTS: Baseline characteristics were balanced across the groups. Only patients receiving 60 and 30mg prasugrel LDs showed a rapid (<1h) and significant (p<0.001) platelet inhibition up to 48h after PCI·The primary EP was met by G3 (p<0.0001), but also G2 scored different (p<0-001) from G4 at 4h after PCI. Similar findings were observed with Verify-Now. No differences in 30-day clinical outcomes were observed across groups. CONCLUSIONS: Switching NSTE-ACS patients before PCI to prasugrel 60 or 30mg LDs determined a better and faster platelet inhibition than continuing clopidogrel, while PCI it is still underway.

Effects of pH and concentration of sodium citrate anticoagulant on platelet aggregation measured by light transmission aggregometry induced by adenosine diphosphate.

The 2013 ISTH-SSC guidelines for the standardization of light transmission aggregometry (LTA) were largely based on expert consensus, as studies directly comparing LTA methodologies were lacking. We experimentally tested the cogency of ISTH-SSC recommendations pertaining to use of anticoagulant, in particular whether: (1) buffered citrate (BC) is preferable to unbuffered citrate (C); (2) the two recommended concentrations of sodium citrate (109 and 129 mM) are equivalent in terms of platelet aggregation (PA). Blood from 16 healthy volunteers was collected into BC and C (109 and 129 mM). PA was measured by LTA in platelet-rich plasma (PRP) stimulated by adenosine diphosphate (ADP) (2 µM) immediately after PRP preparation and up to 4 hr after blood collection; pH and platelet counts in PRP were measured in parallel. pH in PRP increased with time up to about 8 for all anticoagulants, although it was lower in BC than in C at all times. In BC, PA was lower at 45 min, but equivalent at all other times. PA was higher and more stable in sodium citrate 109 mM than in 129 mM at all times. The extent of PA did not change for up to 2 hr after blood collection, and subsequently dramatically decreased. In contrast with ISTH-SSC recommendations, (1) BC does not show advantages compared to C; (2) 109 mM citrate is preferable to 129 mM, because it better supports PA; and (3) LTA studies should be completed within 2 hr of blood collection, instead of the recommended 4 hr.

Reduced platelet count, but no major platelet function abnormalities, are associated with loss-of-function ATP-binding cassette-1 gene mutations.

Loss-of-function mutations of the the ATP-binding cassette-1 (ABCA1) gene are the cause of Tangier disease (TD) in homozygous subjects and familial HDL deficiency (FHD) in heterozygous subjects. These disorders are characterized by reduced plasma HDL-cholesterol (HDL-C) and altered efflux of cholesterol from cells. Previous studies in TD patients and ABCA1-/- murine models reported defects in platelet count, morphology, and function, but the issue is still controversial. We analyzed three subjects with low to very low HDL-C levels due to the loss-of-function mutations of the ABCA1 gene. Two related patients with FHD were heterozygous carriers of two mutations on the same ABCA1 allele; one, with TD, was homozygous for a different mutation. Mild to moderate thrombocytopenia was observed in all the patients. No morphological platelet abnormalities were detected under optical or EM. History of moderate bleeding tendency was recorded only in one of the FHD patients. Only limited alterations in platelet aggregation and activation of the integrin αIIbß3 were observed in one FHD patient. While α-granule secretion (P-selectin), content, and secretion of platelet δ-granules (serotonin, ATP, and ADP) and thromboxane (TX) A2 synthesis were normal in all the patients, the expression of lysosomal CD63, in response to some agonists, was reduced in TD patients. In conclusion, three patients carrying ABCA1 genetic variants had low platelet count, with the lowest values observed in TD, not associated with major alterations in platelet morphology and response to agonists or bleeding.

Inhibition of the platelet P2Y12 receptor for adenosine diphosphate does not impair the capacity of platelet to synthesize thromboxane A2.

AIMS: Patients with acute coronary syndromes (ACSs) are treated with acetylsalicylic acid (ASA) and antagonists of the P2Y12 receptor (P2Y12R) for adenosine diphosphate (ADP). Based on the demonstration that P2Y12R antagonists inhibit thromboxane A2 (TxA2) production (target of ASA), it was surmised that ACS patients might be treated with P2Y12R antagonists only. However, this demonstration contrasts with the results of previous studies. The aim of this study was to test whether P2Y12R antagonists have off-target/indirect inhibitory effects on platelet TxA2 production. METHODS AND RESULTS: We studied 3 patients with inherited P2Y12R deficiency and 33 healthy subjects. Serum TxB2 (TxA2 metabolite) levels were similar in P2Y12R-deficient patients and healthy subjects and were not decreased by P2Y12R antagonists in vitro. Serum TxB2 levels did not decrease in 20 patients treated with prasugrel (10 mg q.i.d.) or placebo for 14 days. Arachidonic acid- and collagen-induced platelet aggregation (PA) and TxB2 production in platelet-rich plasma (PRP) of healthy subjects were inhibited in vitro by P2Y12R antagonists. However, P2Y12R antagonists did not inhibit TxB2 production when PA was prevented by avoiding the stirring of PRP in the aggregometer. The P2Y1 ADP-receptor antagonist MRS2500 had similar effects on PA and TxB2 production as P2Y12R antagonists. Acetylsalicylic acid inhibited TxB2 production more effectively than a P2Y12R antagonist; only the combination of ASA and a P2Y12R antagonist inhibited PA induced by high concentration of collagen. CONCLUSION: Inherited deficiency or pharmacological inhibition of P2Y12R does not affect the platelet capacity to synthesize TxA2. There is no pharmacological evidence that ACS patients may be safely treated with P2Y12R antagonists without ASA.

Congenital defects of platelet function.

Congenital abnormalities of platelet function disorder (PFD) are associated with the heightened risk for bleeding. Typically, patients with PFDs have mucocutaneous bleeding of variable severity and excessive hemorrhage after surgery or trauma. The diagnostic laboratory assessment appropriate for the evaluation of suspected inherited PFD should be based on a two-step diagnostic strategy: the first step, based on screening tests, helps raising a diagnostic hypothesis, which should then be tested in the second step, which is based on the use of specific tests. The first step should include: complete blood cell count, examination of the peripheral blood smear, and assessment of platelet aggregation. Although light transmission aggregometry (LTA) is the most widely used platelet function test, it is relatively insensitive to defects of platelet secretion; for this reason, laboratory tests that measure platelet aggregation and secretion simultaneously, such as lumi-aggregometry, should be preferred to traditional LTA. The second step includes specific tests (e.g., flow cytometry, Western blotting, DNA analysis). Platelet transfusions should be used only to treat severe bleeding episodes. Recombinant Factor VIIa can be used in patients with severe bleeding episodes who do not respond to platelet transfusion because of alloimmunization. Fibrinolytic inhibitors or the vasopressin analogue desmopressin (DDAVP) should be used in all other circumstances.

The platelet count in EDTA-anticoagulated blood from patients with thrombocytopenia may be underestimated when measured in routine laboratories.

Spuriously low platelet counts (PCs) can be observed in normal blood samples anticoagulated with ethylenediamine tetra-acetic acid (EDTA)and, much less frequently, with citrate-tris-pyridossalphosphate (CPT),due to time-dependent in vitro platelet agglutination. Accuracy in PC determination is essential as PC is one of the parameters that usually guides treatment for thrombocytopenic patients. PCs of 93 thrombocy to penic patients were measured in EDTA- or CPT-anticoagulated blood samples immediately after sampling (t0) and 90 min (t90) after storage at room temperature. The presence of platelet agglutinates in blood samples was determined by examining blood smears using optical microscopy.PCs decreased at t90 with both anticoagulants. Platelet agglutinates were present at t90 in 27% of EDTA-samples vs. 2% of CPT-samples with decreased PCs (P < 0.001). Based on PCs in EDTA-samples, 15 patients (16%) shifted from a lower bleeding risk at t0 to a higher bleeding risk category at t90 (P 5 0.019), compared to 5 (5%) patients, based on PCs in CPT-samples. Therefore, time-dependent in vitro platelet agglutination in EDTA-blood samples may cause underestimation of PCs in thrombocytopenic patients, possibly leading to improper management.

Comparison of different procedures to prepare platelet-rich plasma for studies of platelet aggregation by light transmission aggregometry.

Light transmission aggregometry (LTA), the gold standard for the study of patients with defects of platelet function, is a poorly standardized technique. The guidelines that have been produced so far are largely based on consensus of experts, due to the absence of studies directly comparing different procedures. Therefore, ad hoc studies are needed to gather scientific evidence on how to choose the most appropriate procedures for LTA measurement. In this study, we aimed at evaluating the most appropriate conditions for preparing samples of platelet-rich plasma (PRP) for studies of platelet aggregation by LTA. Citrate-anticoagulated blood from 32 individuals was centrifuged at 150, 200, 250 or 300×g at room temperature for 10 min. Red blood cells contamination was highest in PRP prepared at 150×g; mean platelet volume (MPV) was lowest in PRP prepared at 300×g. The extent of platelet aggregation measured by LTA was lower and more variable in PRP prepared at 300×g. Therefore, centrifugation of blood at 200×g or 250×g for 10 min appears to be the best condition for preparing PRP for LTA studies.

Quantification of N-acetyl- and N-glycolylneuraminic acids by a stable isotope dilution assay using high-performance liquid chromatography-tandem mass spectrometry.

The present report describes a method for the quantification of N-acetyl- and N-glycolylneuraminic acids without any derivatization, using their (13)C(3)-isotopologues as internal standards and a C(18) reversed-phase column modified by decylboronic acid which allows for the first time a complete chromatographic separation between the two analytes. The method is based on high-performance liquid chromatographic coupled with electrospray ion-trap mass spectrometry. The limit of quantification of the method is 0.1mg/L (2.0ng on column) for both analytes. The calibration curves are linear for both sialic acids over the range of 0.1-80mg/L (2.0-1600ng on column) with a correlation coefficient greater than 0.997. The proposed method was applied to the quantitative determination of sialic acids released from fetuin as a model of glycoproteins.