In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

To investigate whether altered megakaryocyte morphology contributes to reduced platelet production in idiopathic thrombocytopenic purpura (ITP), ultrastructural analysis of megakaryocytes was performed in 11 ITP patients. Ultrastructural abnormalities compatible with (para-)apoptosis were present in 78% ؎ 14% of ITP megakaryocytes, which could be reversed by in vivo treatment with prednisone and intravenous immunoglobulin. Immunohistochemistry of bone marrow biopsies of ITP patients with extensive apoptosis showed an increased number of megakaryocytes with activated caspase-3 com- IntroductionIdiopathic, or immune, thrombocytopenic purpura (ITP) is an autoimmune disease characterized by isolated thrombocytopenia in an otherwise healthy person. The thrombocytopenia in ITP is caused by accelerated platelet destruction due to the action of antiplatelet immunoglobulin G (IgG) autoantibodies that bind to antigens on the platelet cell membrane. The platelets are subsequently destroyed by tissue macrophages, predominantly in the spleen. 1 As a result of the accelerated destruction, platelet survival is usually greatly shortened and platelet production is thought to be compensatorily increased. 2,3 However, there is also evidence that platelet production can be impaired in ITP. This was demonstrated in platelet kinetic studies using radiolabeled platelets. [4][5][6] The reduced platelet production rate might be mediated by the action of antiplatelet antibodies, which can bind to megakaryocytes in the bone marrow. [7][8][9] Recent in vitro studies support this concept showing that human megakaryocyte colony formation and proplatelet formation is inhibited 10 and that a reduced expansion of megakaryocytic progenitors can be observed especially in the presence of certain antiplatelet glycoprotein antibodies. 11 However, despite the evidence of a reduced platelet production in several ITP patients, numbers of megakaryocytes in the bone marrow are usually normal or increased. 6 This is compatible with the finding that plasma thrombopoietin (TPO) levels in ITP patients are not significantly different from healthy controls, indicating that the total megakaryocytic mass has not been changed in ITP. Investigating the relationship between thrombokinetic parameters and the glycocalicin index (GCI), a parameter of platelet destruction, 12 we recently demonstrated that there is an inverse correlation between the platelet production rate and the GCI. 13 These results suggest that despite the normal number of megakaryocytes in the bone marrow an increased destruction of platelets and/or megakaryocytes might occur. These findings support the concept of ineffective thrombopoiesis in the bone marrow. To investigate whether apoptosis or other forms of programmed cell death are responsible for this ineffective thrombopoiesis, we examined the ultrastructure of bone marrow megakaryocytes from ITP patients with electron microscopy. The results demonstrate that, independent of the refractoriness of ITP to therapy, in all patients most bone ma...

Platelet production requires compartmentalized caspase activation within megakaryocytes. This eventually results in platelet release in conjunction with apoptosis of the remaining megakaryocyte. Recent studies have indicated that in low-risk myelodysplastic syndromes (MDS) and idiopathic thrombocytopenic purpura (ITP), premature cell death of megakaryocytes may contribute to thrombocytopenia. Different cell death patterns have been identified in megakaryocytes in these disorders. Growing evidence suggests that, besides apoptosis, necrosis and autophagic cell death, may also be programmed. Therefore, programmed cell death (PCD) can be classified in apoptosis, a caspase-dependent process, apoptosis-like, autophagic and necrosis-like PCD, which are predominantly caspase-independent processes. In MDS, megakaryocytes show features of necrosis-like PCD, whereas ITP megakaryocytes demonstrate predominantly characteristics of apoptosis-like PCD (para-apoptosis). Triggers for these death pathways are largely unknown. In MDS, the interaction of Fas/Fas-ligand might be of importance, whereas in ITP antiplatelet autoantibodies recognizing common antigens on megakaryocytes and platelets might be involved. These findings illustrate that cellular death pathways in megakaryocytes are recruited in both physiological and pathological settings, and that different forms of cell death can occur in the same cell depending on the stimulus and the cellular context. Elucidation of the underlying mechanisms might lead to novel therapeutic interventions.

To investigate underlying mechanisms of thrombocytopenia in myelodysplastic syndrome (MDS), radiolabeled platelet studies were performed in 30 MDS patients with platelet counts less than 100 ؋ 10 9 /L. Furthermore, plasma thrombopoietin and glycocalicin index (a parameter of platelet or megakaryocyte destruction) were determined. Mean platelet life (MPL), corrected for the degree of thrombocytopenia, was reduced in 15 of 30 patients (4.3 ؎ 0.9 days [mean ؎ SD] vs 6.0 ؎ 1.3, P ‫؍‬ .0003). Platelet production rate (PPR) was reduced in 25 of 30 patients (68 ؎ 34 ؋ 10 9 /d vs 220 ؎ 65, P < .0001). Thrombopoietin levels were not significantly correlated with the PPR. However, the glycocalicin index was significantly higher compared with controls (15 ؎ 16 vs 0.7 ؎ 0.2, P ‫؍‬ .001) and significantly correlated with the PPR (P ‫؍‬ .02, r ‫؍‬ ؊0.5), but not with the MPL (P ‫؍‬ 1 IntroductionMyelodysplastic syndromes (MDSs) are clonal hematologic disorders characterized by ineffective hematopoiesis resulting in one or more cytopenias in the peripheral blood, despite a normo-or hypercellular bone marrow. Anemia is the most common cytopenia, however thrombocytopenia occurs frequently whereby bleeding complications are often noticed. 1,2 Thrombocytopenia in MDS is caused mainly by ineffective platelet production due to a disturbed proliferation and differentiation process of megakaryocytes or their precursors. The cause of the altered megakaryocytopoiesis in MDS has not been elucidated, but recent studies indicate that an increased apoptosis of early progenitors and inhibitory effects of cytokines might contribute to this process. [3][4][5][6][7] However, thrombocytopenia in MDS might also be related to other causes such as concurrent bone marrow fibrosis 8 and immune-mediated mechanisms. [9][10][11] This notion is supported by findings that immunomodulatory agents, such as danazol, antithymocyte globulin (ATG), and cyclosporin A, are effective in some patients with MDS with thrombocytopenia. [12][13][14][15] Furthermore, a reduced platelet life span, measured with radiolabeled platelets, has been found in about 15% of MDS patients with thrombocytopenia. Some of these patients had a recovery of their platelet count after splenectomy. 16 To discriminate further between an increased destruction of platelets in the peripheral blood and an impaired production by the bone marrow megakaryocytes, plasma levels of glycocalicin (GC) and thrombopoietin (TPO) and ultrastructural study of megakaryocytes may be of relevance.GC is a fragment of the platelet membrane glycoprotein Ib. The glycocalicin index (GCI), the plasma GC level corrected for the platelet count, is considered to be a parameter of peripheral platelet turnover. 17 Recently, it was demonstrated in a subgroup of patients with idiopathic thrombocytopenic purpura (ITP) that a decreased platelet production rate, determined with platelet kinetic studies, was associated with an elevated GCI, reflecting an increased destruction of platelets or megakaryocytes in the bone marro...