For quantitative assessment we used cumulative subtraction11 to determine the percentage of CD110+ events relative to an isotype control in a univariate population comparison. for a swift diagnosis on a molecular level. It seems obvious to use this method in the diagnosis of CAMT, which is characterized by a MPL defect. The applicability of this method in the diagnosis of inherited thrombocytopenia and its interference from external influences has not yet been evaluated. Furthermore, the poor availability of monoclonal antibodies in the past hindered its usage for diagnostic purposes in hematological laboratories: over the intervening years none of the commercially available monoclonal antibodies intended for the detection of MPL were found to be suitable, since they proved not to be specific for MPL.9,10 Anguizole We used the monoclonal CD110 antibody clone 1.610 for flow cytometric detection of MPL on platelets and bone marrow cells from both healthy donors and patients with different forms of thrombocytopenia. Peripheral blood and bone marrow from healthy controls and from thrombocytopenic patients were obtained after informed consent was given in accordance with the Declaration of Helsinki. The study has been approved by the ethics committee of the Hannover Medical School. For experimental details see figure legends. For quantitative assessment we used cumulative subtraction11 to determine the percentage of CD110+ events relative to an isotype control in a univariate population comparison. It should be noted that Anguizole percentage positive is a calculated parameter which is affected by both percentage of positive cells and expression level, and has been chosen as a robust parameter which is relatively insensitive to slight variations in instrument settings and experimental conditions. In initial experiments we could demonstrate the expression of MPL on platelets from healthy donors as well as from patients suffering from thrombocytopenia of different etiologies other than CAMT (e.g. idiopathic thrombocytopenia, autosomal dominant macrothrombocytopenia, Wiskott-Aldrich Syndrome). In contrast, but as expected, MPL either could not or could scarcely be measured on the surface of platelets from CAMT patients (Figure 1A). Open in a separate LAG3 window Figure 1. Detection of CD110 (MPL) on platelets. (A) Detection of MPL on platelets: Platelet rich plasma was prepared from anti-coagulated peripheral blood. After short pre-incubation with human immunoglobulins (Gammagard, Baxter Healthcare Corporation, IL, USA) in order to block Fc receptors platelets were stained and analyzed on a flow cytometer (BD FACSCalibur, BD biosciences, San Jose, CA, USA). Histograms show the results from PE-fluorescence from platelets gated on scatter properties. Gray shaded histograms: isotype control (IgG2b-PE, Caltag Medsystems, UK); open histograms: anti CD110-PE (clone 1.6.1, BD biosciences, San Jose, CA, USA). ND: normal donor, XLT: x-linked thrombocytopenia, FA: Fanconi anemia. (B) MPL expression on platelets from thrombocytopenic and non-thrombocytopenic individuals calculated by cumulative subtraction11 was plotted against THPO plasma levels as determined by ELISA (Quantikine, R&D Systems, MN, USA). Data corresponding to THPO concentrations below and above the detection range of the ELISA system were grouped left or right of the gray bars, respectively. We extended our screening to patients with an as yet undiagnosed hypomegakaryocytic thrombocytopenia Anguizole which was suspicious for CAMT. Surprisingly, in a couple of patients we found a wild-type gene despite a complete lack of MPL expression on platelets. Some of these turned out to have another form of congenital hypomegakaryocytic thrombocytopenia or pancytopenia like Fanconi anemia (Figure 1A). Since THPO plasma levels are extremely high in all hypomegakaryocytic forms of thrombocytopenia (up to several ng/ml in contrast to 30 pg/ml in healthy donors), we hypothesized that the lack of MPL expression was due to high THPO levels. Indeed, MPL was detectable neither on platelets from a patient with Anguizole chemotherapy induced severe thrombocytopenia, nor on platelets from a healthy donor 24 h after transfusion to an aplastic anemia patient (synthesis of MPL on early hematopoietic progenitors in contrast to irreversible internalization on platelets. This is in line with experiments showing a recycling of THPO receptor molecules to the cell surface after internalization in a murine hematopoietic cell line but not in platelets.15 The results obtained with more mature CD34+CD38hi progenitors Anguizole lay between those from early progenitors and platelets, revealing a progressive loss of the capability to re-express the receptor.