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Background: Besides essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) the myeloproliferative neoplasms (MPN) defined by the World Health Organization (WHO) comprise the entity of unclassifiable MPNs (MPN, U). The exact differential diagnosis of the specific MPN entities can be challenging particularly at early stages of the diseases. So far, pathologists have had to rely only on histomorphological evaluation of bone marrow biopsies in combination with laboratory data because helpful ancillary tests are not yet available. Even molecular tests, such as JAK2 mutation analysis are not helpful particularly in the differential diagnosis of ET and PMF because both entities are associated with the V617F mutation in 50 % of the cases. Recently overexpression of the transcription factor NF-E2 in MPN was described., Materials and Methods: A collective of samples consisting of 163 bone marrow biopsies including 139 MPN cases was stained immunohistochemically for NF-E2 and analyzed regarding the subcellular localization of NF-E2 in erythroid progenitor cells. The results were compared between the MPN entities as well as the controls and statistical analyses were conducted., Results and Discussion: This study showed that NF-E2 immunohistochemistry and analysis of the proportion of nuclear positive erythroblasts of all erythroid precursor cells can help to distinguish between ET and PMF even in early stages of the diseases. An MPN, U case showing a proportion of more than 20 % nuclear positive erythroblasts can be classified as a PMF with 92 % accuracy.

Bone marrow diagnostics is an essential tool in routine hematological clinical practice. Not only conventional cytological and histological approaches but also more modern techniques, such as immunohistochemistry, cytogenetics and molecular diagnostics are used. The molecular basis of more and more hematological disorders is being discovered and makes its way not only into routine diagnostics but also into daily clinical practice. Recurrent genomic aberrations associated with the individual patient prognosis are well characterized and are being applied in differential therapeutic decisions. In addition, understanding deregulated biochemical pathways have led to the development of targeted therapeutic approaches. This review outlines the value of bone marrow diagnostics in hematological diseases with a focus on the currently emerging molecular diagnostic possibilities.

Thrombocythemia in the course of chronic myeloproliferative disorders like chronic idiopathic myelofibrosis (cIMF) and of course essential thrombocythemia (ET), are characterized by life-threatening complications. In a number of clinical trials the recently introduced drug Agrylin((R)) has proven to be very effective. The normalization of the platelet count was related to an interference with megakaryocyte maturation leading to a left-shifting of this cell lineage and/or a reduced proliferation. However, until now no systematic study has been performed on the relationship between development of megakaryopoiesis and proliferative activity. In this investigation we included 10 patients with cIMF and 5 patients with ET that had received Agrylin((R)) for a period ranging between 6 and 70 months. Following therapy this cohort revealed a decrease in the platelet count from 1,104x10(9)/l at diagnosis to 485x10(9)/l. In this context we focused on an immunohistochemical and morphometric analysis of the CD61(+) megakaryopoiesis involving also endomitotic reduplication, by applying a double-immune incubation technique with the proliferating cell nuclear antigen (PCNA). Moreover, we determined the changes of fiber density during observation time. According to our results, the thrombocytopenic effect of Agrylin((R)) is based on an arrest in the dynamics of megakaryocyte maturation towards large (mature) platelet-shedding (polyploid) cells. This pathomechanism causes a significant increase in the number of promegakaryoblasts and megakaryoblasts. On the other hand, the total amount of CD61(+) megakaryocytic cells is not increased. Related to the peculiar cell biology of endomitotic reduplication during the maturation process and its different periods alloted to each single step, PCNA activity (late G1- and S-phase of the cell cycle) is found to be enhanced in the megakaryocyte precursors. Finally, no significant influence of Agrylin((R)) on the evolution of myelofibrosis is detectable and there is a general improvement of hematological data especially in cIMF.

Differentiation of an elevated, repeatedly determined platelet count (> or =500x10(9)/l) includes the discrimination between reactive causes generated by a variety of underlying conditions and a neoplastic myeloproliferative disorder (CMPD). In addition to clinical findings, the evolution of laboratory data during follow-up and histology of the bone marrow exerts a significant diagnostic impact. Characteristic features are not only expressed by hematopoiesis, but also by the myeloid stromal compartment. While the megakaryocyte-rich subtype of chronic myeloid leukemia (CML) and the 5q(-) syndrome (MDS) are dominated by abnormal micromegakaryocytes, in polycythemia vera (PV) this cell lineage reveals a pleomorphous appearance. In essential thrombocythemia (ET), a prevalence of giant megakaryocytes with deeply lobulated (staghorn-like) nuclei may be encountered. A clear-cut discrimination of ET from early (hypercellular) stages of idiopathic (primary) myelofibrosis (IMF) presenting with thrombocythemia becomes possible, provided the conspicuous atypical features of megakaryopoiesis characterizing the latter entity are taken into account. Moreover, CML displays a predominance of the granulocytic lineage whereas PV shows a panmyelosis or trilineage proliferation, involving erythropoiesis, in particular. In contrast, erythropoiesis is markedly reduced in CML and to a lesser degree also in IMF. In CMPDs extreme values of iron deposits may be found, ranging from a total lack (PV) to minor amounts (CML) and a normal staining reaction (ET). Similar results are exhibited regarding reticulin fibrosis, which is usually not present in ET, rarely observed in PV and detectable to a variable degree in CML and IMF.

Apoptosis and proliferation are important regulators of normal development and homeostasis in the bone marrow. Therefore, dynamics of hematopoiesis is mainly defined by these two parameters. However, since only few data are available from previous studies, we performed a retrospective analysis to elucidate some aspects of this complex pathomechanism. A total of 400 patients with chronic myeloproliferative disorders (CMPDs) and corresponding reactive bone marrow lesions were enrolled into this study. Apoptosis was detected in bone marrow tissue by the ISEL-technique and topoisomerase II alpha expression was demonstrated by the monoclonal antibody Ki-S1. Furthermore, by determination of the proliferating-cell nuclear antigen labeling (PCNA) index, we were able to calculate the proportion of cells in the G2/M phase, because both nuclear antigens are expressed in different phases of the cell cycle. Patients with IMF, PV, and ET revealed a normal range of apoptosis, whereas in chronic myeloid leukemia (CML) a significant increase could be observed. On the other hand, IMF and PV were characterized by a raised proliferative activity. Dynamics of hematopoiesis was assessed by calculation of the so called hematopoietic turnover index. In CML and reactive lesions no alterations of this parameter were detectable, but IMF and PV showed a significant increase. Survival analysis disclosed a relevant worsening of life expectancy for patients with reduced apoptosis and proliferation. In conclusion, our in-situ results confirm and extend previous experimental data on hematopoietic cell kinetics. In this context, a greater regenerative capacity of hematopoiesis may be reflected by an increased rate of apoptosis and/or proliferation and therefore is associated with a more favorable outcome.

A patient with CMGM developed after traumatic splenectomy a severe thrombocytosis up to 4 millions. 25 days after the accident he died of acute right heart failure caused by vascular occlusion of lung vessels due to thrombocyte aggregations. According to the regulations of professional cooperatives in Germany the death is caused by the occupational accident. A bereaved pension will be payed. For the private life insurance the blood disease is with 33% partial responsible for the fatal development and reduces the payment. According to german penal law the death of the victim will tighten the punishment, if someone else is guilty for the accident. A professional error on the part of a doctor is not provable.

We report a case of venous thrombosis presented as ischemic intracranial expanding lesion localized in the parietal lobe of a young man with essential thrombocytosis. As surgical intervention became necessary we were able to investigate the morphological changes by histology and immunocytochemistry. The almost exclusive venous manifestation of thrombosis most probably initiated by elevated amounts of platelets suggests altered blood flow conditions as the predominant factor. Surgical interventions in such conditions are rare and might only proceed in space-occupying lesions with clinical symptoms. Treatment with inhibitors of platelet aggregation under strict control of the patient is the therapy of choice.

60 patients with essential thrombocythemia (ET) have been retrospectively and prospectively followed from 1974 through 1987. The presenting signs and symptoms and the course of the disease were analyzed and compared to 10 patients with persisting reactive thrombocytosis selected from 6,000 patients with reactive thrombocytosis and to 50 patients with other myeloproliferative diseases. 54 ET-patients presented with complications, 46 with thrombembolic, 3 with hemorrhagic problems and 5 with thrombembolic and hemorrhagic problems. In 6 patients ET was detected accidentally. Disturbances of the microcirculation, mainly of the fingers and the toes, were the most frequent symptom. The average maximal platelet count was 1,207,000/microliter. The average platelet count at diagnosis was 880,000/microliter. 16 patients had an elevation of the serum creatinine at diagnosis, which deteriorated during the course of the disease. Bone marrow examinations were performed in 56 patients, histology in 48 patients, cytology in 29 patients. In contrast to the clinical diagnosis the histological diagnosis was in 4 cases each polycythemia vera and myeloproliferative syndrome without further specification. 12 patients died thus far. The causes of death were thrombembolic complications in 9, acute leukemia in 2 patients, in 1 patient the cause of death is not known. 10 years after diagnosis 61% of the patients are still alive. It appears that ET is a more important risk factor for the disturbances of the micro- and macrocirculation than has been recognized until now. ET is, if thrombembolic complications are avoided, a disease with a relatively benign course.