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The iron uptake in bone marrow and spleen was measured in 29 patients with myelofibrosis using 52Fe and quantitative scanning. In 10 patients, no iron uptake in the marrow could be observed and active erythropoiesis was extramedullary only. In the bone marrow of patients with myelofibrosis, the iron uptake per nucleated red cell was less than that observed in conditions without myelofibrosis or extramedullary erythropoiesis. Increasing splenic iron uptake was likely to be associated with a decreasing bone marrow iron uptake and was related to the size of the spleen. The data suggest that in myelofibrosis, the spleen dominates iron uptake through intense erythropoiesis and a high splenic blood flow, thus restraining iron supply to the bone marrow. more...

In order to evaluate a method permitting quantitation of splenic red blood cell destruction, a model of erythrocyte destruction in enlarged spleens was created: Erythrocytes are destroyed in the splenic erythrocyte pool at a constant rate, producing in labelling studies hyperhaemolysis due to random destruction. A mathematical analysis of the model shows that the splenic destruction rate can be calculated with great accuracy from quantitation of the initial excess radioactivity, measured over the spleen during the first days after infusion of 51-Cr-labelled autologous erythrocytes. 18 patients with splenomegaly (479-4700 g) were investigated. The splenic erythrocyte destruction rate was estimated to be between 0.5-4.4% of the total erythrocyte mass per day, increasing significantly with increasing splenic weight. The results indicate that erythrocyte destruction takes place almost exclusively in the enlarged spleen in cases of predominant splenomegaly without complicating immunohaemolysis. more...

33 patients with chronic myelo-proliferative disease were investigated for megakaryocytes in cubital venous blood. 6 Patients had chronic myeloid leukaemia, 11 polycythaemia vera and 16 myelofibrosis. In all patients, a significantly higher number of megakaryocytes than in normal adult humans was found. In patients with chronic myeloid leukaemia and myelofibrosis, an increased number of intact megakaryocytes was found, indicating thrombopoiesis in the peripheral part of the upper extremity. 2 patients with myelofibrosis showed a % of intact megakaryocytes greater than 25, as found in blood from the inferior vena cava in normal adults. In patients with chronic myeloid leukaemia, some of the megakaryocytes were smaller than normal, whereas in myelofibrosis, some of the megakaryocytes were larger than normal, with increased lobulation of nuclei. Active thrombopoiesis in the peripheral part of the extremities can be of great importance, when splenectomy is considered in treatment of myelofibrosis. more...

The hypothesis that transcobalamin I (TC I) originates from granulocytes was tested by comparing the isoelectric focusing (IEF) patterns of the R-type binder (cobalophilin) of vitamin B12 of: 1) lysed granulocytes, 2) 24 h granulocyte output, and 3) plasma. The preparations came from the blood of 5 normal subjects and 4 with myeloproliferative states. The cobalophilin released into a culture of granulocytes resembled the binder in the granulocytes initially. TC I, defined as the alpha1 cobalophilin with components isoelectric between pH 2.9-3.35 and as a carrier of native plasma B12, was not released by granulocytes. The granulocyte binder of leukaemic granulocytes did not differ from the normal in content per cell or amount released. more...

Elevated levels of platelet-associated IgG and/or IgM were found in 15 of 18 patients with myelofibrosis (83%). All but 5 patients with elevated PAIg had active disease. The amounts of PAIg were not correlated to either S-Ig, platelet count or spleen size. Levels of PAIgG well above the normal range were especially found in patients with short duration of disease and/or a transitional myeloproliferative state. It is debated whether immune-mediated platelet dysfunction may be of importance for the development of bone marrow fibrosis, mediated by the release of platelet-derived growth factors in the bone marrow. Elevated PAIg may also contribute to abnormal haemostasis and thrombocytopenia in myelofibrosis. more...

3 patients with myelofibrosis with myeloid metaplasia were splenectomized because of anaemia and disturbing splenomegaly. In the course of the 6 months following splenectomy, a polycythaemia developed. Erythrokinetic studies demonstrated that in all cases a reduction in plasma volume and an increase in red cell volume was obtained. Total erythropoiesis decreased along with normalization of ineffective erythropoiesis and peripheral haemolysis. The reappearance of an erythropoietic activity measured over the sacrum was a constant finding, while in 1 patient, a depression of activity over the liver was observed. The new distribution and organization of erythropoiesis in the splenectomized patients is hypothesized as being due to the removal of the influence of an enlarged spleen on erythropoietic organs. more...

Morphometric evaluation was performed on semi-thin sections of core biopsies of the bone marrow and included 20 cases of each group of diseases besides control specimens. (i) Hyperergic myelitis of rheumatic origin. (ii) Chronic granulocytic leukaemia (CGL). (iii) Polycythaemia vera (P. vera). (iv) Chronic megakaryocytic-granulocytic myelosis (CMGM). (v) Myelofibrosis or osteomyelosclerosis (MF/OMS). The following classification of megakaryopoiesis was applied: normal megakaryocytes; giant forms; microforms; intussusceptions; cytoplasmic fragments; naked nuclei. The density distribution shows an increase of megakaryocyte number in those 5 different marrow disorders, ranging from about 13/mm2 in the normal sample up to 65 cells/mm2 in MF/OMS. Microforms are most frequently encountered in CGL, whereas giant megakaryocytes, intussusceptions and many cytoplasmic fragments characterize P. vera, CMGM and MF/OMS. Our measurements suggests 3 distinct categories of bone marrow lesions with corresponding alterations of the megakaryopoiesis: (i) myelitis and CGL; (ii) P. vera; (iii) CMGM and MF/OMS. more...

2 cases of megakaryocytic leukaemia are presented as a variant of myeloproliferative disorders. The uncontrolled proliferation of the megakaryocyte-platelet cell line occurred after splenectomy and treatment of blastic phase. Applicability of the term megakaryocytic leukaemia is discussed as well as its relationship to similar disorders. It is suggested that an extensive neoplastic proliferation of megakaryocytes with abnormal maturation and thereby formation of large dysplastic platelets in extramedullary organs especially the liver in the end stage of myelofibrosis is a possible mechanism for this disorder. Treatment with platelet-pheresis may be an effective part of therapy. more...

The circulating GM-CFC density-distribution profile was studied in 19 cases of myelofibrosis with myeloid metaplasia. The distribution profile for the population studied appeared to be similar to that of normal bone marrow GM-CFC's but shifted toward lower densities in comparison with normal circulating GM-CFC's. For individual patients, it appeared that the greater the circulating blood GM-CFC's concentration, the lower the mean density. It appears likely that the premature release into blood of the GM-CFC's in myelofibrosis is a property of the neoplastic haematopoietic stem cell clone at the core of the disease. more...

17 patients with idiopathic myelofibrosis were studied for the occurrence of circulating immune complexes (IC), using a polyethylene glycol complement consumption and a polyclonal rheumatoid factor inhibition assay. In 13 patients complement C3d was determined by rocket immunoelectrophoresis. Circulating IC were detected in 6 patients and were primarily found in patients with short duration of disease from time of diagnosis. The median duration of the disease in IC-positive patients was 4 months, compared to 12 months in the IC-negative group (P less than 0.05). 9 of the 13 patients investigated had increased levels of plasma C3d. However, there was no correlation to the occurrence of IC. It is concluded that circulating IC may take part in an immune-mediated bone marrow damage. This may involve deposition of IC in the bone marrow with secondary inflammation responsible for the development of bone marrow fibrosis. more...

Granulocyte-committed progenitor cells (colony-forming units, CFUc) in the blood of 10 patients with primary myelosclerosis and 2 patients with myelosclerosis following polycythaemia vera were assayed by the agar culture technique. The mean number of CFUc was 54.1 +/- 109 (SD) (range 1.4--394) x 10(6)/1 which corresponded to an increase of more than 1000-fold above normal levels. There was a linear relationship of CFUc with total leucocyte count in the different patients. The magnitude of this increase resembles that found in untreated patients with chronic granulocytic leukaemia and is therefore in keeping with the concept that the concentration of CFUc in the circulation is due to a primary increase in their number and not to disordered release from the marrow. more...

A case of typical myelofibrosis with a huge spleen is described in a 62-year old man. During the subsequent 2-year follow-up, a clinical picture of polycythaemia vera with pancytosis and disappearance of the marrow fibrosis was observed. The pancytosis necessitated treatment with busulphan and frequent phlebotomies. The transformation was associated with prednisone treatment for a suspected haemolytic state. During this treatment, the spleen no longer became enlarged on clinical palpation, although it was still enlarged at post mortem examination, but much less than 2 years earlier. more...

Using a radioimmunoassay the serum concentration of the N-terminal propeptide of type III procollagen (P-III-P) was measured in 35 patients with chronic myeloproliferative disorders, including idiopathic myelofibrosis (n = 10), osteomyelosclerosis (n = 4), transitional myeloproliferative disorder (n = 5), polycythaemia vera (n = 10) and chronic myelogenous leukaemia (n = 6). The normal range in 35 healthy controls was 4.9-11.7 ng/ml. The serum concentration of P-III-P increased with increasing degrees of bone marrow reticulin fibrosis. By contrast, almost normal levels were detected in osteomyelosclerosis with an indolent clinical course, in which an excessive deposition of mature collagen fibres was found, representing mainly type I collagen. These observations indicate that the serum P-III-P level is positively correlated to the degree of bone marrow reticulin fibrosis, whereas levels are near normal in patients with osteomyelosclerosis and stable disease. Measurement of serum P-III-P may be a useful indicator of disease activity in myelofibrosing conditions. more...

In 9 patients with myeloproliferative diseases (MPD) (6 with myelofibrosis, MF, 1 with Ph1 positive chronic granulocytic leukaemia, CGL, 1 with primary eosinophilia, PE, 1 with pre-leukaemia syndrome, preL) collagen, epinephrine, and ADP-induced aggregation, N-ethylmaleimide-induced malondialdehyde (MDA) production, beta-thromboglobulin (beta-TG) plasma levels, and platelet turnover were studied. Collagen-induced aggregation was found to be normal in 7 patients, absent in 1, and reduced in 1. In all but 3 patients, aggregation with ADP was markedly reduced. Epinephrine-induced aggregation was decreased in 7 patients. No difference was found between mean MDA production in MPD (3.21 +/- 0.50 nmol/10(9) PLTs) and in control group of 21 normal subjects (3.04 +/- 0.26 nmol/10(9) PLTs). Mean beta-TG levels were significantly higher (P less than 0.01) in MPD patients (165.00 +/- 28.29 ng/ml) than in healthy controls (81.76 +/- 14.63 ng/ml). Mean platelet production half-time was significantly shorter in MPD (2.48 +/- 0.24 d) than in the control group (3.43 +/- 0.17 d), after adjustment for age by covariance analysis (P less than 0.005). Our data do not indicate an abnormal prostaglandin synthesis and are consistent with the hypothesis that a disseminated intravascular platelet aggregation might take place in MPD patients. more...

In response to specific treatment of vitamin B12 deficient, anaemic patients there is an influx of folate into the young, circulating red cells. To separate new and old cells, capillary tubes filled with whole blood were centrifuged and the packed red cell column divided into top (T), middle (M) and bottom (B) layer. The newest cells are found in the T layer. The increase in red cell folate (RCF) concentration starts before, during or after the reticulocyte response, and is therefore not directly related to folate metabolism in the red precursor cells in the marrow. The low RCF concentration at the peak of the reticulocyte response in some of the cases demonstrates that the folate material, which may have been accumulated in the red precursor cells in the marrow, may be lost by the time the red cells enter the peripheral blood. The influx of folate into the young, circulating red cells is rapidly followed by an efflux of folate, suggesting that much of the folate material is still in the monoglutamate form. A new influx of folate is noted after a time lapse of from 5 to 10 d. Iron deficiency seems to prevent the uptake of folate by the circulating red cells. more...

Platelets of patients with myeloproliferative disorders (MD) such as polycythaemia vera (PV), chronic myelogenous leukaemia (CML), idiopathic myelofibrosis (IM) and essential thrombocythaemia (ET) have been found to have low 5HT levels measured both by a fluorimetric and a liquid chromatographic assay. Km and Vmax for platelet active uptake of 3H-5HT were not significantly different in controls and patients. Inhibition of 5HT reuptake by imipramine or induction of moderate release by fenfluramine were not sufficient to distinguish the group of MD platelets from controls, although some patients had less of a tendency to retain intraplatelet amine. The low platelet 5HT content found in our patients seems not to be the consequence of disturbed active transport of 5HT across platelet membrane. Although defective storage of this amine within the cell is probable, the results of the present study do not rule out the possibility that platelets from MD patients undergo in vivo activation by endogenous stimuli not inhibited by aspirin. 10 d treatment with aspirin did not result in any significant rise in intraplatelet 5HT concentration. more...

Using an enzyme-linked immunosorbent assay (ELISA), elevated RBC-Ig of the IgG and IgM class were found in 8 of 14 patients with idiopathic myelofibrosis. In 2 patients with high levels of RBC-Ig the direct Coombs' test was positive. It is supposed that immune haemolysis may contribute to the anaemia in some patients with idiopathic myelofibrosis. more...

A 67-year-old woman investigated because of 'myelodysplastic syndrome' was found to have a 4-fold increase in G-6-PD activity in her erythrocytes. The enzyme was partially purified and characterized. On grounds of: (a) reduced electrophoretic mobility, (b) abnormal cathodic band(s) in isoelectrofocusing, (c) increased Michaelis constant for glucose 6-phosphate, (d) abnormal thermostability, and (e) abnormal interaction with the ligand NADPH, we conclude that this is a new structural variant which we designate G-6-PD Verona. G-6-PD Verona was the sole apparent source of G-6-PD activity in the patient's erythrocytes; by contrast, the patient's fibroblasts had only normal G-6-PH (type B). The patient's haematological course terminated into acute myeloid leukaemia. We believe G-6-PD Verona was the result of a somatic mutation in an X-chromosome which took place in a haemopoietic cell clone which subsequently underwent neoplastic transformation. more...

Marrow fibrosis is involved in some haematological malignancies. Either because of sampling errors, variations of focal distribution of fibrosis or the discomfort for patients of bone biopsies, conventional histology appears to be unsuitable for the follow-up of myelofibrosis. During collagen synthesis by marrow fibroblasts, the aminoterminal propeptide is removed from procollagen III and released in the serum. Thus, a sensitive radioimmunoassay of type III aminoterminal propeptide of procollagen (PC III) has been tested in myeloproliferative and lymphoproliferative disorders with a marked bone marrow fibrosis. In polycythaemia vera, PC III level was significantly increased as compared to controls and was related to marrow fibrosis of grade I. The more increased PC III values were observed in spent polycythaemia cases initially treated by phlebotomy alone. Follow-up showed a transformation into myeloid metaplasia. In contrast, PC III remained stable in patients treated with radiophosphorus 32P or hydroxyurea who did not transform. In myeloid metaplasia, results of PC III were significantly higher than in controls or polycythaemia vera cases. Myelofibrosis of recent onset (less than 2 years) gave higher values than chronic myelofibrosis. Increased PC III values were also emphasized in chronic myelocytic leukaemia, and in a few cases of refractory anaemia with excess of blasts, hairy cell leukaemia and chronic lymphocytic leukaemia. more...

Quantitative and qualitative changes in granulocyte-macrophage (CFU-GM) and fibroblast colony-forming cells (CFU-F) were studied in 7 patients with primary myelofibrosis (MF). Marrow cells were collected from bone biopsy specimens after treatment with collagenase. The number of CFU-GM correlated with the amount of haemopoietic tissue noted in the bone marrow histology and ranged between 0-400/mg of bone. CFU-F were increased in 2 patients with moderate fibrosis. Circulating CFU-GM were increased in all patients studied (169-3749/ml of blood). There was no significant correlation between the number of CFU-GM in the bone marrow and that in the blood. Cytochemical studies showed a high incidence in eosinophil progenitors in the bone marrow and especially in the blood of patients with MF. These data suggest a functional abnormality of myeloid progenitors in this disease. more...