Enumeration and immunohistochemical characterisation of bone marrow basophils in myeloproliferative disorders using the basophil specific monoclonal antibody 2D7

BACKGROUND: Basophils are highly specialised granulocytes that express a unique profile of antigens and increase in myeloproliferative disorders (MPD). In chronic myeloid leukaemia (CML), basophilia is a diagnostic and prognostic determinant. So far, however, no reliable approach for routine detection and enumeration of bone marrow basophils has become available. OBJECTIVE: To detect and enumerate basophils in bone marrow sections in patients with CML and other MPD. METHODS: The anti-basophil antibody 2D7 was applied to paraffin embedded bone marrow sections from normal/reactive subjects (n = 31), patients with CML (chronic phase, n = 37; accelerated phase, n = 9), and other MPD (chronic idiopathic myelofibrosis (CIMF), n = 20; polycythaemia vera (PV), n = 20; essential thrombocythaemia (ET), n = 20; indolent systemic mastocytosis (ISM), n = 7). RESULTS: As assessed by serial section staining, 2D7(+) cells were found to co-express myeloperoxidase, histidine decarboxylase, CD9, and CD43, but did not express B cell or T cell restricted antigens. 2D7(+) bone marrow cells were found to increase in CML compared with normal/reactive bone marrow and other MPD (median numbers of 2D7(+) cells/mm(2): CML, 33; normal/reactive bone marrow, 6; CIMF, 10; PV, 6; ET, 5; ISM, 3; p<0.05). The highest basophil counts were recorded in accelerated phase CML (115/mm(2)). CONCLUSIONS: A novel immunohistochemical procedure has been established for basophil detection in normal bone marrow and MPD. This approach should help in the quantification of bone marrow basophils at diagnosis and during anti-leukaemic treatment.     

Mast cell burden and reticulin fibrosis in the myeloproliferative neoplasms: A computer-assisted image analysis study

The mast cell has been associated with fibrosis in many different tissues, organs, and different disease processes including hematopoietic malignancies. Mast cells are often increased in the bone marrow of patients with primary bone marrow disorders, and patients with systemic mastocytosis often have a second concomitant neoplastic disease of the bone marrow. The goals of the current study were to determine the role the mast cell has in the pathogenesis of myeloproliferative neoplasms (MPN) and to correlate the mast cell burden with the degree of reticulin fibrosis.We used computer-assisted image analysis of bone marrow core biopsies stained for mast cell tryptase from patients with myeloproliferative neoplasms [31 cases: 12 chronic myelogenous leukemia (CML), 6 primary myelofibrosis (PMF), 4 essential thrombocythemia (ET), 4 polycythemia vera (PV), and 5 chronic myeloproliferative disorder, unclassifiable (CMPD-U)].Although the number of cases of some subtypes of MPN was small, the results suggested that PMF and ET each had significantly more mast cells than both CML and control cases (P<0.01 and 0.05, respectively, Mann–Whitney test). CMPD-U and PV showed no significant differences from the control cases, but the CML cases had significantly fewer mast cells than our control cases (P=0.02, Mann–Whitney test). In addition, the quantity of mast cells seen in the bone marrows of MPN patients correlated with reticulin fibrosis (P=0.04, Mann–Whitney test).Our studies highlight the different mast cell quantities in different myeloproliferative neoplasms and suggest a direct role for the mast cell in intramedullary fibrosis. Further studies are warranted to confirm our observation and to study the mechanisms by which mast cells contribute to fibrosis in the MPN setting.     

Thrombopoietin receptor (Mpl) expression by megakaryocytes in myeloproliferative disorders

The thrombopoietin receptor (Mpl) is involved in the pathogenesis of chronic myeloproliferative disorders (CMPD). In this study, we determined Mpl expression by bone marrow cells and megakaryocytes in CMPD by applying laser microdissection, real-time RT-PCR, and immunohistochemistry. Mpl mRNA expression was significantly increased up to 9-fold in total bone marrow cells (p < 0.001) and up to 4-fold in megakaryocytes in chronic myeloproliferative disorders (n = 73) compared to normal controls (n = 26, p = 0.01). Immunohistochemistry revealed heterogeneous Mpl expression by megakaryocytes in CMPD with a stronger accentuation in idiopathic myelofibrosis (IMF) in comparison to polycythaemia vera (PV) and essential thrombocythemia (ET). In addition to megakaryocytes, the erythropoietic lineage was prominently labelled by Mpl antiserum, with considerably stronger staining in polycythaemia vera. We conclude that, in CMPD, megakaryocytes and erythroid cells exhibit increased Mpl expression levels which may contribute to the sustained proliferation of both cell lineages in CMPD.     

QBEND10 for the diagnosis of myelodysplastic syndromes in routinely processed bone marrow biopsy specimens.

AIM--The assessment of the value of the antibody QBEND10, which is directed against the haemopoietic stem cell related antigen CD34, in the immunohistochemical diagnosis of myelodysplastic syndrome in routinely processed bone marrow biopsy specimens. METHODS--581 formalin fixed, paraffin embedded trephine biopsy specimens of the iliac crest were immunostained with QBEND10 (avidin-biotin complex/ABC method). The number of CD34+ haemopoietic stem cells/blast cells (referred to hereafter as CD34+ cells) was determined in each case. The Wilcoxon test was used for statistical analysis. RESULTS--The following diagnostic categories were defined: (1) normal or reactive bone marrow (n = 356), (2) lymphoproliferative disorders, usually non-Hodgkin's lymphoma of low grade malignancy or multiple myeloma (n = 118), (3) myelodysplastic syndrome (n = 22), (4) acute leukaemia (n = 44), and (5) myeloproliferative diseases (n = 41). The average number of CD34+ cells was very low (0.2/HPF) in normal and reactive bone marrow, in lymphoproliferative disorders and in the myelodysplastic syndrome subtypes RA and RARS. Myeloproliferative diseases showed an average of three CD34+ cells/HPF. However, the average number of CD34+ cells was significantly higher (p < 0.05) in the myelodysplastic syndrome subtypes RAEB and RAEB-T (8.7/HPF) and in acute leukaemia (including both myeloid and lymphoblastic leukaemia; 111.7/HPF). CONCLUSIONS--QBEND10 is of value for the identification of RAEB and RAEB-T in routinely processed bone marrow biopsy specimens because it enables the detection of even small increases in the number of CD34+ cells.     

骨髓增殖性疾病Bcr／Abl融合基因的表达及其意义

目的：为探讨几种骨髓增殖性疾病bcr／abl 融合基因出现的频率及临床意义．方法：采用逆转录多聚酶键反应（RT－PCR）技术,研究了慢性粒细胞白血病（CML）,真性红细胞增多症（PV）,原发性血小板增多症（ET）,原发性骨髓纤维化（MF）bcr／abl基因的变化．结果：48例慢性期CML,43例呈现bcr／abl基因阳性,占90％,14例加速,急变期CML,6例出现bcr／abl基因,阳性率为43％,与慢性CML相比P＜0.01;10例PV出现1例bcr／abl基因阳性,占10％,6例ET中未发出bcr／abl基因．2例MF中发现1例bcr／abl基因阳性．结论：bcr／abl基因检测对于CML的临床分型有重要意义,bcr／abl阴性的CML预后差,而bcr／abl基因的阳性有助于初诊时的CML急变与急性非淋巴细胞白血病（AML）M2的鉴别和CML与慢性粒单细地白血病（CMML）的鉴别．在其它三种骨髓增殖性疾病中MF可能与CML关系最为密切,而ET关系较远,少数PV可能转化为CML．     

Chronic myeloproliferative disorders: the role of tyrosine kinases in pathogenesis, diagnosis and therapy.

The term chronic myeloproliferative disorders was originally used by Damashek to describe the link amongst a group of acquired blood diseases. Recent molecular genetic analysis has provided a scientific basis for this observation. Underlying myeloproliferative disorders are acquired abnormalities of tyrosine kinase genes. These may be chromosomal translocations resulting in the creation of a fusion kinase gene, examples of which include ABL, FGFR, and PDGFR as seen in disorders CML, 8p11 myeloproliferative syndrome, atypical CML and chronic eosinophilic leukaemia. The second group of tyrosine kinase abnormalities are point mutations in JAK2, a cytosolic TK. This abnormality is seen in 30-97% of cases of MPD with the phenotype PV, ET or CIMF.     

Bone marrow stroma in idiopathic myelofibrosis and other haematological diseases. An immunohistochemical study

Bone marrow stroma was investigated immunohistochemically in 31 patients with haematological diseases, mainly idiopathic myelofibrosis (n = 8) and related chronic myeloproliferative disorders (n = 14). The bone marrow from patients with idiopathic myelofibrosis and some CML patients showed marked staining reactions with antibodies against type III procollagen (pN collagen), type IV collagen, fragment P1 of laminin and factor VIII. Patients with osteomyelosclerosis had particularly increased collagen content, including both newly deposited type III collagen (pN collagen) and mature collagen fibres. As in normal bone marrow, argyrophilic fibres and type III collagen displayed a close co-distribution, which was also demonstrated for type IV collagen and laminin. While normal bone marrow sinusoids had discontinuous basement membranes, fibrosing bone marrow was characterized by endothelial cell proliferation and capillarization, with the development of continuous sheets of basement membrane material beneath endothelial cells.     

Current diagnostic criteria for the chronic myeloproliferative disorders (MPD) essential thrombocythemia (ET), polycythemia vera (PV) and chronic idiopathic myelofibrosis (CIMF)

The clinical criteria for the diagnosis of essential thrombocythemia (ET) according to the polycythemia vera study group (PVSG) do not distinguish between ET and thrombocythemia associated with early stage PV and prefibrotic chronic idiopathic myelofibrosis (CIMF). The clinical criteria of the PVSG for the diagnosis of polycythemia vera (PV) only detects advanced stage of PV with increased red cell mass. The bone marrow criteria of the World Health Organization (WHO) are defined by pathologists to explicitly define the pathological criteria for the diagnostic differentiation of ET, PV, and prefibrotic and fibrotic CIMF. As the clinical PVSG and the pathological WHO criteria show significant shortcomings, an updated set of European Clinical and Pathological (ECP) criteria combined with currently available biological and molecular markers are proposed to much better distinct true ET from early PV mimicking ET, to distinguish ET from thrombocythemia associated with prefibrotic CIMF, and to define the various clinical and pathological stages of PV and CIMF that has important therapeutic and prognostic implications. Comparing the finding of clustered giant abnormal megakaryocytes in a representative bone marrow as a diagnostic clue to MPD, the sensitivity for the diagnosis of MPD associated with splanchnic vein thrombosis was 63% for increased red cell mass, 52% for low serum EPO level, 72% for EEC, and 74% for splenomegaly indicating the superiority of bone marrow histopathology to detect masked early and overt MPD in this setting. The majority of PV and about half of the ET patients have spontaneous EEC, low serum EPO levels and PRV-1 over-expression and are JAK2 V617F positive. The positive predictive value for the diagnosis of PV of spontaneous growth of endogenous erythroid colonies (EEC) of peripheral blood (PB) and bone marrow (BM) cells is about 80-85% when either PB or BM EEC assays, and up to 94% when BM and PB EEC assays were performed. The diagnostic impact of low serum EPO levels (ELISA assay) in a large study of 186 patients below the normal range (<3.3 IU/l) had a sensitivity specificity and positive predictive value of 87%, 97% and 97.8%, respectively, for the diagnosis of PV. There is a significant overlap of serum EPO levels in PV versus control and controls versus SE. The specificity of a JAK2 V617F PCR test for the diagnosis of MPD is high (near 100%), but only half of ET and MF (50%) and the majority of PV (up to 97%) are JAK2 V617F positive. The use of biological markers including JAK2 V617 PCR test, serum EPO, PRV-1, EEC, leukocyte alkaline phosphatase score and peripheral blood parameters combined with bone marrow histopathology has a high sensitivity and specificity (almost 100%) to diagnose the early and overt stages of ET, PV and CIMF in JAK2 V617F positive and negative MPDs.     

Mutation analysis of ASXL1, CBL, DNMT3A, IDH1, IDH2, JAK2, MPL, NF1, SF3B1, SUZ12, and TET2 in myeloproliferative neoplasms

Since the discovery of the JAK2V617F tyrosine kinase-activating mutation several genes have been found mutated in nonchronic myeloid leukemia (CML) myeloproliferative neoplasms (MPNs), which mainly comprise three subtypes of "classic" MPNs; polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). We searched for mutations in ASXL1, CBL, DNMT3A, IDH1, IDH2, JAK2, MPL, NF1, SF3B1, SUZ12, and TET2 genes in 149 non-CML MPNs, including 127 "classic" MPNs cases. JAK2 was mutated in 100% PV, 66% ET and 68% MF. We found a high incidence of ASXL1 mutation in MF patients (20%) and a low incidence in PV (7%) and ET (4%) patients. Mutations in the other genes were rare (CBL, DNMT3A, IDH2, MPL, SF3B1, SUZ12, NF1) or absent (IDH1).     

Evaluating the volume ratio of bone marrow affected by fibrosis: a parameter crucial for the prognostic significance of marrow fibrosis in chronic myeloid leukemia

Marrow fibrosis (MF) is a complication of bone marrow neoplasms that usually impairs quality of life and shortens survival time. Proof and exact quantification of MF is not yet standardized, thus impeding the comparability of results and the evaluation of its prognostic impact. In this study on 360 bone marrow biopsy specimens from 135 patients with either chronic idiopathic myelofibrosis (CIMF) (evaluation group) or chronic myeloid leukemia (CML) (test group), marked differences were detected between six different approaches systematically compared with respect to proof and quantification of MF. A new volumetric approach quantifying the marrow volume affected by fibrosis turned out to be superior to all of the other morphometric methods considering practicability and specificity of results, and superior to a semiquantitative procedure considering sensitivity, precision, and reproducibility (P < 0.00005). The assessment of the marrow volume with fibrosis was the only feature of MF independently influencing the survival time of patients (test group with CML; multivariate analysis, P = 0.0008). We conclude that an approach estimating the marrow volume affected by fibrosis is the method of choice to exactly quantify and prove MF. The loss of marrow volume due to fibrosis appears to be crucial with respect to the prognostic significance of MF in CML. Hum Pathol 34:391-401.     

Osteoclasts and bone remodeling in chronic myeloproliferative disorders. A histochemical and morphometric study on trephine biopsies in 165 patients

In 165 patients with chronic myeloproliferative disorders (CMPD) a morphometric and histochemical study was performed on trephine biopsies of the bone marrow to elucidate osseous remodeling by assessment of trabecular bone area (planimetry) and number of osteoclasts. Osteoclastic elements were identified by the tartrate-resistant acid phosphatase method. In addition to control specimens (n = 20) subtypes of CMPD included chronic myeloid leukemia (CML, n = 65), primary (essential) thrombocythemia (PTH, n = 25), polycythemia vera rubra (P. vera, n = 25) and agnogenic myeloid metaplasia (AMM, n = 50). AMM was discriminated into a so-called early hyperplastic stage without gross myelofibrosis (n = 19) and an overt or advanced stage showing fibro-osteosclerotic changes (n = 31). Total area of trabecular bone and counts for osteoclasts (uni- and multi-nucleated cells as well as a-nuclear cytoplasmic fragments) were not significantly increased in CML, PTH, P. vera and in the initial hypercellular stages of AMM. In contrast to these results, in advanced stages of AMM there was a significant increase in total bone area associated with a high count for all osteoclastic elements and apparently also an increased number of osteoblasts. It is speculated that the marked increase in osteoclastic-osteoblastic elements in late stages of AMM possibly reflects an imbalance of calcitriol (1.25-dihydroxyvitamin D 3) on skeletal homeostasis. This abnormal osseous remodeling may be mediated by the atypical megakaryocytic proliferation in this disorder, which is always a conspicuous feature of bone marrow biopsies.     

Thrombozytose versus Thrombozythämie – zur Differentialdiagnose des erhöhten Plättchenwertes

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.     

Cytogenetics and molecular genetics in myelofibrosis with myeloid metaplasia and polycythemia vera

Myelofibrosis with myeloid metaplasia (MMM) is a rare myeloproliferative disorder (MPD) characterized by clonal proliferation of hematopoietic progenitors. 40-50% of karyotypes on blood (or more rarely on bone marrow) revealed at least one abnormality: 30% at diagnosis and 90% in blastic transformation phase. A minority of patients with newly diagnosed polycythemia vera (PV) presented chromosomal abnormalities in their myeloid cells. The most frequent visible alteration in MMM and PV is a 20q deletion, also characterized in other MPDs and myeloid malignancies. Among other chromosomal changes, deletion 13q is more common in MMM than in other MPDs, trisomy 9 and 9p alterations appear more frequent in PV. Cytogenetic studies have disclosed cryptic anomalies and pointed out the high frequency of 9p alterations. JAK2 (V617F) mutation was found in almost all PV patients and near half of MMM patients. This molecular abnormality takes an increased importance in the knowledge of the physiopathology of MPDs, particularly in PV and also in prognosis of MMM patients.     

Differences in CD33 intensity between various myeloid neoplasms

We measured the concentration of CD33 antigen on the surface of cells in 315 bone marrow (BM) samples and 114 corresponding peripheral blood (PB) samples from patients with various leukemias (acute myeloid leukemia [AML], chronic myelogenous leukemia [CML], myeloproliferative disorder [MPD] other than CML, myelodysplastic syndrome [MDS]) and from control subjects. Overall CD33 intensity in total CD33+ cells was significantly higher in BM than in PB. CD33 intensity in total BM CD33+ cells differed significantly with the type of disease. The median number of CD33 molecules per cell was highest in AML, followed by MDS, CML, and control subjects and lowest in MPD. When only CD34+/CD33+ cells were examined, CD33 molecules per cell were highest in CD34+ cells in AML and lowest in MPD (P = .027). Patients with AML or MDS younger than 60 years had significantly higher intensity of CD33 expression on CD34+ cells than patients 60 years or older. Levels of CD33 intensity did not correlate with cytogenetics in patients with AML or MDS. There was no correlation between CD33 intensity and response to therapy or overall survival in 35 patients treated with protocols including Mylotarg. These data demonstrate variation in CD33 intensity between various leukemias.     

Enzyme negative blastic transformation of chronic myeloproliferative disorders: immunophenotyping of the blastic cell population.

Enzyme negative blast cells from 27 patients with chronic myeloproliferative disorders (CMPDs) in blastic transformation were analysed with a panel of monoclonal antibodies (MoAbs). According to morphologic features of the bone marrow and laboratory data, the 27 cases were divided into 8 cases of myelofibrosis (MF), 3 cases of chronic megakaryocytic granulocytic myelosis (CMGM) and 16 cases of chronic myeloid leukaemia (CML). Of the 27 cases, 23 showed a positive reaction with myeloid MoAbs, but in 12 cases expressing myeloid markers, megakaryocytic, monocytic or lymphoid cell features were also detected. In 7 cases of MF, 1 case of CMGM and 1 case of CML a bilineage, myelo-megakaryocytoid immunophenotype of peripheral blast cells was seen. Of the 4 patients with CML expressing lymphoid markers, 2 showed early B-cell, 1 T-cell surface antigens, and 1 both myeloid and early B-cell features. In this group of cytochemically immature blastic transformation of CMPD, only 1 case was termed "undifferentiated" blastic transformation.     

Novel system for degeneration of blood vessels by UV irradiation and subsequent regeneration using chick bone marrow cells

Recently, many results have been reported regarding the pluripotency of bone marrow cells (BMCs) with the aim of benefiting regenerative medicine for humans. Particularly, vessel formation by hematopoietic stem cells or vascular endothelial stem cells which were derived from bone marrow has received considerable interest, since the mechanism of vessel formation has been found to be involved in neoangiogenesis of serious diseases such as cancer. Most work on neoangiogenesis and regeneration has involved mammalian experimental systems, however the avian model is useful since the process of neoangiogenesis and regeneration of vessels can be observed with the whole embryo culture system. We have established a novel system using early chick embryos, where a portion of blood vessels are degenerated by UV irradiation, and vessel regeneration is then studied. Incubated embryos were partially covered with aluminum foil, from the embryonic body to the dorsal marginal vein, and irradiated with UV for 1 min. Donor BMCs were obtained from the femurs and tibias of chicks aged 10 days, fluorescently labeled with PKH26 and injected into the anterior vitelline vein of the recipients. In BMC-treated embryos the donor BMCs were observed around the UV-degenerated vessels, and regeneration of blood vessels occurred, in contrast to the untreated embryos. These results indicate that avian BMCs have the ability to participate in vessel regeneration, and the avian model used here may be a useful tool for studies of vessel neoangiogenesis and repair.     

Grading of marrow fibrosis in chronic myeloid leukemia--a comprehensive approach

In the course of Chronic myeloid leukemia (CML), appearance of increased number of blasts may herald evolution of accelerated phase as well as onset of marrow fibrosis (MF) thereby necessitating the need to perform trephine biopsy for correct diagnosis and appropriate treatment. In the existing grading systems of MF, a comprehensive view has not been taken of the variability of density and area occupied by reticulin and collagen fibres. To overcome this shortcoming, we quantitated the reticulin and collagen content of marrow, its pattern of distribution and percentage area occupied by each type of fibres in every individual case. We performed 50 bone marrow (BM) trephine biopsies in patients of CML in order to assess the incidence and degree of MF. Various grades of MF were correlated with peripheral smear including blast count, bone marrow aspirate and LAP score of the case. A positive correlation was found between increasing grades of MF and number of megakaryocytes in the BM.     

Nucleolar organizer regions of megakaryocytes in chronic myeloproliferative disorders

Summary To study megakaryocyte activation, the argyrophilic staining method of nucleolar organizer regions (AgNOR) has been applied to decalcified bone marrow biopsies of 16 individuals with no haematopoietic disorders and 59 patients with chronic myeloproliferative disease. Of the 59 patients, 18 had chronic myeloid leukaemia (CML), 21 chronic megakaryocytic granulocytic myelosis (CMGM), 13 polycythaemia vera (PV) and 7 essential thrombocythaemia (ET). The AgNOR number of megakaryocytes in CML was significantly lower, and in CMGM, PV and ET significantly higher than in healthy individuals. The high number and the clusters of fine-grained AgNORs of megakaryocytes in CMGM, PV and ET are suggestive of active, proliferating cells. The AgNOR number of megakaryocytes and the platelet counts of the patients did not show a convincing correlation. In CMGM, PV and ET the pyknotic, heterochromatinized megakaryocytes with narrow rims of cytoplasm called bare (nude) nuclei, possessed few, large AgNOR granules. The AgNOR staining of bare nuclei and the roughly identical number of granules found in CMGM, PV and ET indicate a common, active mechanism of apoptosis.     

Fibrosis identified in the bone marrow biopsies of patients with essential thrombocythemia: its incidence and significance for the differential diagnostic considerations

Myelofibrosis (MF) may develop in all types of myeloproliferative disorders and its identification is of clinical relevance. Typical bone marrow (BM) morphology of patients with essential thrombocythemia (ET) shows either "normal" amount or "a slight increase" of reticulin fibers, but the published data differ in relation to the applied MF definition and ET diagnostic criterias. The aim of this study was to evaluate retrospectivelly MF in BM biopsies of 30 cases in which the diagnosis of ET was confirmed also clinically by local hematologists. In 7 of the patients not only primary but also sequential biopsy was available. The MF grade and extent were evaluated semiquantitativelly in archival slides stained by G?m?ri silver impregnation. The analysis was based on the European clinicopathological criteria 2004 (ECP) defining a) normal bone marrow fibrosis (MF0), b) slight reticulin fibrosis (MF1), c) advanced reticulin and initial collagen fibrosis (MF2) and d) advanced collagen fibrosis (MF3). Generally, in majority of the biopsies MF0 (n = 6) or MF1 (n = 25, 18x focal and 7x diffuse) was found. More advanced MF2 was much less common as it was present in 6 biopsies (5x focal and 1x diffuse). In relation to the actual time of BM biopsy during course of the disease, the introductory biopsies done at the time of diagnosis (n = 18) showed 3x MF0, 14x MF1 and 1x MF2. The biopsies performed after a long time of patients observations (n = 12) showed 3x MF0, 7x MF1 and 2x MF2. In 5 of 7 sequential biopsies the progress of MF was evident, but 4 of these patients were treated by cytoreductive therapy. We conclude that the BM of patients with ET in initial phase shows either MF0 or focal slight increase of reticulin fibers (MF1). In addition, the long course of the disease and/or applied therapy may lead to more developed MF and more advanced MF stages (diffuse MF1 or MF2). Therefore their finding in the BM biopsies examined in the later phases of the disease should not exclude the diagnosis of ET.