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Despite the publication in 2009 of a paper on 'terms and definitions of immune thrombocytopenia' (ITP), some unresolved issues remain and are reflected by the disagreement in the treatment suggested for primary ITP in adults. Considering that these disagreements could be ascribed to non-shared goals, we generated a 'consensus' on some terms, definitions, and assertions useful for classifying the different lines of treatment for primary ITP in adults according to their indications and goals. Agreement on the appropriateness of the single assertions was obtained by consensus for the following indicators: 1 . classification of four 'lines of therapy'; 2 . acceptance of the expression 'sequences of disease' for the indications of the respective four lines of treatment; 3 I . practicability of splenectomy; 3 Ib . acceptance, with only some exceptions, of a 'timing for elective splenectomy of 12 months'; and 4 a-d . 'goals of the four lines of therapy.' On the basis of the consensus, a classification of four lines of treatment for primary ITP in adults was produced. In our opinion, this classification, whose validity is not influenced by the recently published new guidelines of the American Society of Hematology (ASH) and reviews, could reduce the disagreement that still exists regarding the treatment of the disease.

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 A 2 -independent PA was tested in all subjects. Blood samples were collected in citrate (C) [low Ca 2+ ] or lepirudin (L) [physiological Ca 2+ ]; platelet count was adjusted to 250 x 10 9 /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.

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 P2Y 12 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.

The gray platelet syndrome (GPS) is a rare congenital platelet disorder characterized by mild to moderate bleeding diathesis, macrothrombocytopenia and lack of azurophilic α-granules in platelets. Some platelet and megakaryocyte (MK) abnormalities have been described, but confirmative studies of the defects in larger patient cohorts have not been undertaken. We studied platelet function and bone marrow (BM) features in five GPS patients with NBEAL2 autosomal recessive mutations from four unrelated families. In 3/3 patients, we observed a defect in platelet responses to protease-activated receptor (PAR)1-activating peptide as the most consistent finding, either isolated or combined to defective responses to other agonists. A reduction of PAR1 receptors with normal expression of major glycoproteins on the platelet surface was also found. Thrombin-induced fibrinogen binding to platelets was severely impaired in 2/2 patients. In 4/4 patients, the BM biopsy showed fibrosis (grade 2-3) and extensive emperipolesis, with many (36-65%) MKs containing 2-4 leukocytes engulfed within the cytoplasm. Reduced immunolabeling for platelet factor 4 together with normal immunolabeling for CD63 in MKs of two patients demonstrated that GPS MKs display an alpha granule-specific defect. Increased immunolabeling for P-selectin and decreased immunolabeling for PAR1, PAR4 and c-MPL were also observed in MKs of two patients. Marked emperipolesis, specific defect of MK alpha-granule content and defect of PAR1-mediated platelet responses are present in all GPS patients that we could study in detail. These results help to further characterize the disease.

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.

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.

Objective: The percentage measurement of hypochromic red cells (HYPO) and reticulocyte haemoglobin content (CHr) using the ADVIA system has recently been validated as a useful tool in indicating iron deficiency, also in cases of chronic diseases such as renal failure. The aim of this study was to evaluate the extent to which the red cell parameters, RBC-Y and RET-Y, provided by Sysmex XE 2100, correlate with HYPO and CHr., Material and Methods: The laboratory markers of iron status were evaluated together with HYPO, CHr, RBC-Y and RET-Y in 92 healthy subjects (C), 42 iron-deficient patients (ID) and 88 uraemic patients receiving regular dialysis treatment (RDT)., Results: In ID patients, increased HYPO and decreased RBC-Y, CHr and RET-Y values, with no overlapping with reference values, were found and a significant correlation was present between ADVIA 120 and Sysmex indices (p<0.001 for each correlation). In RDT patients, HYPO median values were increased with a wide distribution of values (95 % reference range = 0.7-27.5 % and 0.7-22.6 % in men and women, respectively). In contrast, RBC-Y was normal/increased (95 % reference range = 169.4-191.1 and 168.7-190.5 in men and women, respectively), even though there was a significant correlation between them (p<0.001). CHr and RET-Y values were within the reference range; moreover, in these patients mean cell volume of red cells and of reticulocytes (MCV and MCVr) median values were increased., Conclusions: This study confirmed the validity of RBC-Y in the management of ID, but not in RDT, where the diagnostic power of RBC-Y as an index of cell hypochromia is limited owing to high MCV values.