Characteristics of rat megakaryocyte colonies and their progenitors in agar culture.

The characteristics of megakaryocyte colonies that develop from megakaryocyte progenitors of rat bone marrow stimulated by rat spleen-conditioned medium (SCM) in agar culture were investigated. Colony frequency was optimal on day 7 and increased relative to both the number of cells plated and the concentration of SCM used. Plating efficiencies averaged 72 +/- 16 megakaryocyte colonies/10(5) cells with 0.1 ml SCM/culture. Colonies were categorized as small cell and big cell. Small-cell colonies had a greater proliferative potential, with a mean of 25 cells/colony. Big-cell colonies averaged 15 cells/colony. The ratio of big-cell to small-cell colonies was 0.69 +/- 0.29. Granulocyte-macrophage colonies, which were also stimulated by SCM, accounted for 70% +/- 15% of the total colonies in the cultures. Cytocidal experiments with tritiated thymidine reduced megakaryocyte colony formation by 45% and granulocyte-macrophage colony formation by 21%. The properties of rat, mouse, and human megakaryocyte progenitors as assayed in vitro are compared.

The effects of thrombopoietic activity of rabbit plasma fractions on megakaryocytopoiesis in agar cultures.

A plasma fraction that stimulates platelet production in vivo also stimulates megakaryocytopoiesis in vitro. The plasma activity is attributed to a humoral regulator, thrombopoietin. Addition of the thrombocytopenic plasma (TP) fraction to an agar system supporting megakaryocyte colonies increased the frequency of colony formation significantly over that stimulated by spleen cell-conditioned medium (SCM). TP had no effect on the size of the colonies and, in the absence of SCM, TP did not stimulate colony formation. In studies of single megakaryocytes, the numbers of small megakaryocytes, specifically those 5-10 microns in diameter, increased significantly after 3 days of incubation with TP alone. SCM supported not an increase in the numbers, but an increase in the proportion of larger (30- to 40-microns) megakaryocytes. A normal plasma fraction contained similar but consistently less activity than fractions containing TP. The findings indicated that TP stimulates differentiation of megakaryocyte precursors from unidentifiable to identifiable cells but does not alone support colony formation. Thus, TP appears to be a potentiator of megakaryocytopoiesis. However, the augmentation of colony frequency by TP further suggests that TP may also play a role in early colony development, either by enhancing progenitor responsiveness to a megakaryocyte colony-stimulating factor or by recruiting additional colony progenitors from a noncycling progenitor population. These studies establish a link between the stimulation of platelet production observed after TP administration in vivo and the effects of TP on early events in megakaryocytopoiesis.

Megakaryocytopoiesis in experimentally induced chronic normobaric hypoxia.

It was recently proposed that prolonged hypoxia produces hypomegakaryocytic thrombocytopenia by reducing the pool of committed megakaryocyte progenitor cells at the expense of a greatly expanded erythroid progenitor pool. In order to test this hypothesis we have studied the relationship between megakaryocytopoiesis, erythropoiesis, and granulopoiesis at the level of progenitor cells (megakaryocyte colony-forming unit, CFU-Mk; erythroid CFU, CFU-E; erythroid burst-forming units; BFU-E; and granulocyte-macrophage CFU, CFU-GM) in the marrow of rats exposed for 4 weeks to normobaric hypoxia. We have found that hypomegakaryocytic thrombocytopenia was accompanied by decreased CFU-Mk, increased CFU-E, and a normal number of BFU-E and CFU-GM. These results support the hypothesis that prolonged hypoxia reduces the precursor cell commitment to differentiate into the megakaryocyte series by enhancing demand for differentiation into the erythroid cell line. However, the underlying mechanism needs further investigation.

Erythroid progenitors in anemic Belgrade laboratory (b/b) rats.

The anemia of Belgrade b/b rats has been shown to be due to intracellular iron deficiency. The aim of this study of erythropoiesis at the progenitor cell level in these rats was to determine if a defect is present in the early phase of red cell production. Both erythroid colony-forming unit (CFU-E)- and erythroid burst-forming unit (BFU-E)-derived colonies were found to be few in untreated b/b rats and made up of a small number of poorly hemoglobinized erythroblasts of different size and irregular cell shape. Following treatment with iron, the anemia of the rats improved, and the number of CFU-E-derived colonies and the number of cells per colony increased, but the peculiar erythroblast morphology persisted. The high serum level of biologically active erythropoietin (Ep) in b/b rats rules out inadequate Ep production as a cause of their anemia. The results presented indicate, in addition to the earlier described defective transmembrane iron transport, a defect in erythroid progenitor cells. The effect of iron treatment in these rats detected in vitro on erythroid progenitors confirms the importance of iron for cellular proliferation.

Late thrombosis of the Björk-Shiley tilting disc valve in the tricuspid position. Thrombolytic treatment with streptokinase.

The main complication after implantation of a Björk-Shiley tilting disc valve in the tricuspid position is late thrombotic obstruction. Of 28 patients with tricuspid valve replacement (16 with mitral, aortic, and tricuspid valve replacement; 12 with mitral and tricuspid valve replacement), with a mean follow-up of 5.2 years, seven (25%) had thrombosis of the tricuspid prosthesis. Three patients had a recurrent thrombotic malfunction, for a total of ten thrombotic malfunctions in 146 patient-years, a rate of 6.8 per 100 patient-years. Thrombosis occurred late in all patients. Clinical deterioration presented with signs of congestive heart failure. In all patients the click of the tricuspid prosthesis was not audible and new diastolic or systolic murmurs were. The diagnosis was confirmed with cineradiography and bidimensional echocardiography (immobile disc, diminished opening angle of the disc). Thrombolytic treatment with streptokinase was used in all seven patients. Two patients required 12 hours of therapy and five patients, 24 hours. Thrombolytic treatment was monitored by the thrombin time. Complete regression of clinical, cineradiographic, and echocardiographic signs fo thrombosis was seen in all seven patients during the first 24 hours of therapy. There were no bleeding complications. In one patient clinical signs of mild pulmonary embolism occurred and were confirmed with chest radiographs. Follow-up, after successful treatment, extends from 4 to 30 months (mean 16.5 months). In four patients long-term results are excellent: There have been no clinical, cineradiographic, or echocardiographic signs of rethrombosis of the tricuspid prosthesis during the follow-up. Rethrombosis of the tricuspid prosthesis was observed in three patients 4, 7, and 14 months after initial treatment with streptokinase. Repeat thrombolytic treatment with streptokinase was successful in all three of these patients. Our experience with streptokinase treatment of thrombosis of tricuspid Björk-Shiley prostheses indicates that this form of treatment should always be applied before surgical intervention.

Two karyotypically unrelated clones with the t(5;17) and deletion of 5q in myelodysplastic syndrome.

We report a patient with primary myelodysplastic syndrome (MDS) and two coexisting karyotypically independent clones. Cytogenetic investigation of bone marrow (BM) cells at diagnosis showed, besides the cells with normal karyotype, a clone that manifests an interstitial deletion of the long arm of chromosome 5 and a second one with a t(5;17). The rarity of finding a BM mosaicism in myelodysplasia with 5q- in some cells and different chromosomal abnormalities in others may be considered to support the multistep theory of pathogenesis in MDS.

A case of chronic myeloid leukemia without Ph1 translocation.

A case of chronic myeloid leukemia (CML) with a short survival (11 months) is described. Cytogenetic peculiarities of the bone marrow cells analyzed by G-banding consisted of a Ph1 chromosome with no translocation and a translocation t(5;13). Hematological characteristics were marked leukocytosis and massive splenomegaly. After treatment with busulfan complete hematological remission was achieved, followed by the appearance of a normal clone. However, 6 months later the patient entered the blastic crisis and a hyperdiploid clone appeared. The usual chemotherapy was given, but the patient responded only partially and died with a prevalence of pathologic myeloblasts in the bone marrow, corresponding to progression of the hyperdiploid clone.

Multifocal plasmacytoma of hand and foot bones.

Simultaneous occurrence of localized plasmacytomas of both hands and feet has not been reported so far. Here we report a 40-year old female patient, who had at presentation pain and deformity. Of hands and feet, with numerous cystic lytic lesions of phalangeal, metacarpal and metatarsal bones, detected by X-rays. The biopsy of the affected bone showed moderately differentiated plasmacytoma of lambda light chain type (lambda-LC). Serum and urine biochemical analysis revealed the existence of lambda LC monoclonal component. The patient was treated by local radiotherapy and subsequent systemic chemotherapy, which consisted of 3 cycles of the M-2 protocol and 7 cycles of melphalan-prednisone. Five years after the diagnosis, the absence of plasmacytoma was confirmed by puncture biopsy of the left hand phalanx. Monoclonal protein in serum and urine was not detected.

Myelofibrosis in primary myelodysplastic syndromes: clinical and biological significance.

In a retrospective study of 236 patients with primary myelodysplastic syndromes (MDS), 130 cases (55.1%) revealed myelofibrosis in bone marrow biopsies. It was observed that fibrosis mostly occurs focally or patchy, and collagen deposits were found very rarely (only four patients). The histopathology of bone marrow biopsies revealed several differences between fibrotic and non-fibrotic MDS: cellularity is significantly higher, dysmegakaryopoiesis is more pronounced, plasmocytes and mast cells are more often increased, and disturbance of marrow topography (particularly of the MK- and G-line) can be found more frequently in MDS with myelofibrosis. Reticulin fibrosis occurred in all subtypes of MDS; however, there was a higher incidence in chronic myelomonocytic leukemia. The frequency of abnormal growth of GM-progenitors was significantly higher in the MDS cases with myelofibrosis, compared to the cases without fibrosis. Clinical data showed significantly higher WBC, more frequent presence of immature granulocytes, and higher percentage of myeloblasts in peripheral blood and bone marrow in MDS with myelofibrosis compared to cases without myelofibrosis. Life expectancy was reduced to 13 mo, compared with 35 mo in MDS without fibrosis (p=0.00055). Time to leukemic transformation was 32 mo in MDS with fibrosis, compared with >56 mo in MDS without fibrosis (p=0.015). Myelofibrosis therefore seems to herald a poor prognosis.

Biological and clinical significance of clonogenic assays in patients with myelodysplastic syndromes.

Biological and clinical significance of growth pattern of hematopoietic progenitors were investigated in 117 patients with primary myelodysplastic syndromes (MDSs) at referral. Abnormal (i.e., "leukemic" or absent) growth of GM colonies (CFU-GM) and GM clusters was found in 47% of patients with "advanced" MDS (RAEB, RAEB-t, and CMML) and in 15% of "low-risk" (RA/RARS) patients. In vitro erythropoiesis was decreased in most of the patients, with significantly lower number of BFU-E in "advanced" MDS than in RA/RARS patients. Megakaryocyte progenitors (CFU-MK) were very low or absent in almost all the patients, regardless of the FAB type. Significant correlation was demonstrated between the number of BFU-E and hemoglobin concentration and between number of CFU-MK and platelet count. Growth capacity of GM progenitors appears to be in proportion to "myeloproliferative" capacity of the malignant clone. T-cell depletion had no influence on growth capacity of hematopoietic progenitors, nor did colony growth respond in a dose-dependent manner to different concentrations of LCM. Growth capacity of MDS hematopoietic progenitors was independent of Bournemouth score, of the presence and type of cytogenetic abnormality, and of the expression of CD95 and caspase-3 antigens on bone marrow cells. However, in patients with "abnormal" growth of GM progenitors, CD34 antigen expression was significantly higher than in patients with "normal" growth. "Abnormal" GM growth was found to be independently predictive regarding the survival and the risk for AML development. In contrast, the prognostic value of erythroid and megakaryocyte cultures was found to be limited.

Primary wound closure in haemophiliacs undergoing dental extractions.

215 teeth were extracted in 62 haemophiliacs following a single infusion of factor VIII supplemented with antifibrinolytics. Tooth sockets were packed with Sorbacel gauze soaked with an antifibrinolytic. 1 group was sutured and the other left open. Primary closure of the extraction wound did not show direct effect on haemostasis. However, the size of the clot showed a statistically significant difference between the primary closure and open wound groups. The authors conclude that primary closure of the extraction wound protects the blood clot, makes the postoperative period more comfortable for patients and may subsequently decrease the risk of postoperative bleeding.