Diagnosis of essential thrombocythemia at platelet counts between 400 and 600x10(9)/L. Gruppo Italiano Malattie Mieloproliferative Croniche(GIMMC).

BACKGROUND AND OBJECTIVE: Diagnostic criteria for essential thrombocythemia (ET) remain essentially negative, that is, exclusion of other myeloproliferative diseases and causes of reactive thrombocytosis. A platelet count above 600x10(9)/L is still generally considered an absolute diagnostic criterion although new protocols for positive diagnostic criteria have recently been proposed, reducing the stringency of a definite platelet limit. This study demonstrates that a platelet count 600x10(9)/L is not a reliable diagnostic criterion for ET, especially in the early stages. DESIGN AND METHODS: An ongoing retrospective study by the GIMMC analyzed 2,316 ET patients diagnosed between 1986 and 1995. Of these 2,316 patients, diagnosed according to the PVSG criteria, 68 had a platelet count 600x10(9)/L and were analyzed separately; 37 out of 68 were excluded from this analysis because of a follow-up shorter than 2 years and/or because of treatment with myelosuppressive agents. The remaining 31 patients were the subjects of our study. RESULTS: After a median follow-up of 4.56 years (range 2-9.6 years) none of the 31 patients had a spontaneous decrease of platelets to the normal range. Transformation to a different chronic myeloproliferative disorders was never observed and no patient developed a condition known to produce reactive thrombocytosis. During follow-up, 23 patients (74%) were treated with anti-aggregating drugs, mainly aspirin. The disease did not evolve into acute leukemia in any patient, 1 had a thrombotic event and none presented hemorrhagic episodes. Median platelet count during follow-up was 534x10(9)/L (range 398-997x10(9)/L). INTERPRETATION AND CONCLUSIONS: Long term follow-up has documented that our 31 patients were correctly diagnosed as having ET, although platelet count was 600x10(9)/L. Our patients were probably in a early phase of their disease and following updated PVSG criteria would have been misdiagnosed leading to incomplete recognition of the natural history of the disease. Further, because an early diagnosis could also have a clinical relevance, our results outline the need for new criteria for the diagnosis of ET. The exclusion of patients with a platelet count between 400 and 600x10(9)/L may prevent patients, nevertheless at risk of vascular complications, from being treated.

Interferon-alpha 2b is not effective in the treatment of refractory immune thrombocytopenic purpura.

About 25-30% of patients with immune thrombocytopenic purpura (ITP) are refractory to corticosteroids, splenectomy and other treatments. It has been suggested that interferon-alpha 2b (IFN-alpha 2b) may be useful in the treatment of chronic refractory ITP patients. We treated 9 chronic refractory ITP patients with IFN-alpha 2b: the results were poor.

Effect of PEG-recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on growth and differentiation of the HEL cell line.

BACKGROUND AND OBJECTIVE: Thrombopoietin has been established as the major regulator of megakaryocyte and platelet production. In this study we evaluated the effects of PEG-recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a pegylated and truncated form of thrombopoietin, on the growth and differentiation of the HEL cell line. As a model system we chose the pluripotent HEL line that acquires multiple markers of the megakaryocyte/platelet phenotype following treatment with phorbol esters, and, more importantly, expresses the receptor for thrombopoietin (Mpl receptor) at its cellular surface. DESIGN AND METHODS: The effect of PEG-rHuMGDF on HEL proliferation/differentiation was evaluated in a liquid culture assay. RESULTS: Peg-rHuMGDF do not increase the proliferative capacity of HEL cell but, in parallel experiments, HEL cells showed a more mature and differentiated pattern after exposure to the cytokine. Our results show that PEG-rHuMGDF, at the optimal doses of 100-150 ng/mL, is able to induce: 1) morphological changes with the formation of cytoplasmic protrusions; 2) increased ploidy as demonstrated by cytofluorimetric analysis; 3) increased expression of megakaryocyte markers, including glycoprotein IIb-IIIa and the platelet-specific alloantigen (PIA1). INTERPRETATION AND CONCLUSIONS: These findings show that HEL cells represent a useful model to investigate the differentiative properties of thrombopoietin in the megakaryocyte compartment.

Megakaryocyte progenitors derived from bone marrow or G-CSF-mobilized peripheral blood CD34 cells show a distinct phenotype and responsiveness to interleukin-3 (IL-3) and PEG-recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF).

In the present study we investigated the proliferative response of megakaryocyte progenitor cells (CFU-MK) derived from peripheral blood stem cell (PBSC) collections of patients with haematological malignancies and normal donors. Highly purified CD34+ cells and mononuclear cell fractions were assayed in the presence of recombinant interleukin-3 (IL-3) and pegylated-recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), alone or in combination, and megakaryocyte colony formation was evaluated in the plasma clot. In comparison, steady-state bone marrow samples from normal donors were highly enriched in CD34+ cells and tested with the cytokines studied. Our results showed that IL-3 was able to stimulate CFU-MK colony formation from bone marrow and peripheral blood CD34+ cells. Similarly, PEG-rHuMGDF stimulated, in a dose-response manner, CD34+ cells from the bone marrow. However, normal mobilized peripheral blood CD34+ cells were not induced to generate CFU-MK colonies by PEG-rHuMGDE The same lack of response was observed when patients peripheral blood CD34+ cells primed with chemotherapy plus G-CSF or with G-CSF alone were assessed. In contrast, PEG-rHuMGDF stimulated CFU-MK growth when mononuclear cells, either from the bone marrow or from mobilized peripheral blood, were grown in plasma clot. Moreover, we analysed by flow cytometry the expression of Mpl receptor on the cell membrane of normal mobilized peripheral blood and normal steady-state bone marrow CD34+ cells. Our results showed a reduced expression of Mpl receptor on mobilized peripheral blood progenitor cells in comparison with bone marrow cells.

Percutaneous umbilical blood sampling in the management of immune thrombocytopenic purpura during pregnancy.

Severe neonatal thrombocytopenia occurs in about 15% of deliveries from women with immune thrombocytopenic purpura (ITP). Conflicting data exist about the real usefulness of percutaneous umbilical blood sampling (PUBS) in evaluating the fetal platelet count. We report successful experience, using PUBS, in the management of 12 pregnant women with ITP.

Slimming diet and the development of acute leukemia: a possible relationship? Report of three cases.

Diet may play a key role in the pathogenesis of cancer and evidence for the role of a reduced intake of micronutrients with antioxidant properties have been increasingly reported. Until now, epidemiologic research in humans has focused on the negative effect of different diets containing excessive caloric intake and relatively little is known on the possible correlation between slimming diets and the incidence of acute leukemia. In this report we describe the temporal association of imbalanced slimming regimens and the subsequent diagnosis of acute leukemia in three cases. This association may be coincidental or perhaps suggests a possible relationship of the diet with the development or progression of acute leukemia.

Granulocytic sarcomas: clinical, diagnostic and therapeutical aspects.

Granulocytic sarcomas (GS) are extramedullary tumor masses of immature myeloid cells most frequently associated with acute myeloblastic leukemia. We report our diagnostic, clinic and therapeutic approaches in the treatment of 6 patients affected by GS who presented with different localizations and symptoms: mediastinal mass with chest pain, rectal tumor with bowel occlusion, bladder mass with acute kidney failure, quadriceps tumor with pain, vertebral localization with pain and bowel mass with pain, respectively. The correct diagnosis of GS by bone biopsy, the immunohistological evaluation of the tumor masses, the prompt use of active drugs in the first line therapy schedule as for acute myeloblastic leukemia are the parameters for the achievement of the long-term remission.

Successful treatment of essential thrombocythemia in a pregnancy with recombinant interferon-alpha 2a.

Essential thrombocythemia (ET) in pregnancy is associated with adverse perinatal outcome, which is likely due to thrombosis of the uteroplacental circulation. Obstetrical management is still controversial. Different therapeutic protocols have been adopted, including the use of aspirin, plateletpheresis, and interferon. We herein report a case of ET treated with interferon-2 alpha from 13 weeks gestation until term. Therapy was well tolerated, leading to a linear reduction of platelet and white blood cell count that rapidly returned within normal limits. A healthy, 3,020-g male infant was delivered at 40 weeks gestation. Albeit further experience is required, recombinant interferon-alpha 2a may play an important role in preventing complications in pregnant patients with ET.

Endothelium and bone marrow transplantation.

Thrombotic complications may occur early after marrow transplantation and many data suggest that endothelial injury plays a pivotal role in their pathogenesis. Since plasma thrombomodulin and P-selectin are thought to be of value as markers of vascular endothelial cell membrane injury, we investigated their plasma concentration in bone marrow transplant patients aiming better to clarify the degree of endothelial involvement. Plasma thrombomodulin and P-selectin were monitored in 25 patients without thrombotic complications before transplant, on day 0 and weekly for 1 month thereafter, while in three patients who developed VOD monitoring continued until day +52. These proteins were in the normal range in all the uncomplicated patients and in two with reversible VOD, while they were always very high in the only patient who developed very severe and lethal VOD. In conclusion, we suggest that endothelial activation/damage occurs rarely in the course of BMT for hematological malignancies; we were able to document endothelial injury in only one patient with very severe thrombotic complication.

Increased P-selectin plasma levels in patients with thrombotic thrombocytopenic purpura.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a rare vascular disorder of unknown etiology. There is evidence to support the hypothesis that platelets and endothelium play a pivotal pathogenetic role. Immunological assays for plasma thrombomodulin and P-selectin levels have recently been made available and they allow simple evaluation of endothelial damage and endothelial/platelet activation, respectively. In this study, we measured the plasma levels of thrombomodulin, P-selectin and von Willebrand factor in 9 TTP patients during active disease and at the time of complete remission (CR). METHODS: Thrombomodulin, P-selectin and von Willebrand factor were measured by enzyme immunoassay. RESULTS: Mean thrombomodulin and von Willebrand factor plasma values were always within the normal range. P-selectin plasma levels, both in the active phase of the disease and in CR (median 312 and 185 ng/mL, respectively), were significantly higher than in normal controls (mean 96 +/- 35 ng/mL, mean 88 ng/mL; p < 0.05). However, the mean value of P-selectin in CR (median 185 ng/mL) was significantly lower than that observed at diagnosis (p < 0.05). In addition, an inverse relationship between P-selectin plasma levels and platelet count (r = -0.526; p = 0.03) was observed. CONCLUSIONS: These findings suggest that activation of platelets and/or endothelium may play a relevant role in the pathogenesis of TTP.

Negligible hemostatic toxicity of intermediate-dose Erwinase in adult patients with acute lymphoblastic leukemia: preliminary data.

The hemostatic toxicity of low dose L-asparaginase from Erwinia carotovora (Erwinase) has been reported to be negligible in adult patients with acute lymphoblastic leukemia (ALL); conversely, no consistent data have been obtained when Erwinase is administered at intermediate doses. We report preliminary clinical and laboratory hemostatic data from 10 adult patients with ALL treated during induction phase with intermediate doses of Erwinase (20,000 IU/m2s.c. every other day, for a total of six administrations). No thrombotic or hemorrhagic events were registered and the mean values of PT, aPTT, fibrinogen, antithrombin and D-dimer did not change during treatment. Only one patient showed a decrease of antithrombin (48% on day 8) requiring temporary suspension of Erwinase therapy. These data suggest that intermediate doses of Erwinase also have negligible hemostatic toxicity in adult patients with ALL.

Subconjunctival THC:YAG laser sclerostomy for the treatment of glaucoma: preliminary data.

A subconjunctival thermal sclerostomy was performed using the recently developed THC:YAG laser in 33 eyes with a variety of glaucomas in which, in most cases, neither medical therapy nor previous procedures had been successful in controlling intraocular pressure (IOP). The mean preoperative IOP was 27.5 +/- 7.5 mm Hg with maximum medication. The laser procedure was quick and easy, with minimal manipulation of tissues; complications were clinically insignificant. The day after the operation, 23 eyes had an IOP less than 18 mm Hg; in the other 10, it was unchanged. In most of the eyes, an obvious filtering bleb developed at the site of the sclerostomy. The internal opening of the sclerostomy was visible on gonioscopy, except in five cases, in which it was plugged by a fold of the iris root. One year after the operation, IOP was controlled in three eyes without medication and in 18 with medication. IOP remained uncontrolled in the remaining 12 eyes.

Validation of a HPLC method for the determination of bendazac and its main metabolite 5-hydroxybendazac in human plasma.

The purpose of this study was to validate an analytical method for the determination of bendazac and its main metabolite 5-hydroxybendazac in human plasma. The results obtained indicate that the method is reproducible, accurate, precise, sensitive and specific for the measurement of bendazac and 5-hydroxybendazac in the human plasma. Therefore it can be considered suitable for experimental purposes, routine application for drug monitoring and regulatory requirements.

Validation of a HPLC method for the determination of bendazac and its main metabolite 5-hydroxybendazac in rabbit aqueous humor and its applicability to human aqueous.

The purpose of this study was to validate an analytical method for the determination of bendazac and its main metabolite 5-hydroxybendazac in aqueous humor. The method was validated with rabbit aqueous but it can be used also for human aqueous since no differences between the two matrices were observed. The results obtained indicate that the method is reproducible, accurate, precise, sensitive and specific for the measurement of bendazac and 5-hydroxybendazac in the aqueous humor. Therefore it can be considered suitable for experimental purposes, drug monitoring and adequate for regulatory requirements.

Evaluation of Keeler Pulsair non-contact tonometry: reliability and reproducibility.

Intraocular pressure was evaluated in 414 eyes using Keeler Pulsair tonometry in comparison with values obtained by Goldmann tonometry. No statistically significant differences were found between the two techniques. Landis K statistical analysis was used to evaluate the measurements obtained by two operators in 125 eyes, with the results indicating good reproducibility. In 20 eyes, a statistically significant difference was found between values obtained with patients in the seated vs supine positions, whereas measurements made with subjects' heads in a supported or unsupported position yielded no statistically different values.

Evaluation of the 701 interzeag lens opacity meter.

The Interzeag 701 Lens Opacity Meter was used to study the lenses of 122 cataract eyes and 73 healthy eyes with clear lenses. The readings taken separately in 67 eyes by two different operators did not significantly differ. Statistically significant differences were found between the readings taken by the same operator when the pupil size was different in all types of cataract and also in eyes with transparent lenses. Opacity Meter readings correlate well with the visual acuity of eyes with nuclear and mixed cataracts, but no correlation was found in eyes with cortical or posterior subcapsular cataracts. In healthy eyes with clear lens, a correlation was found between the instrument readings and age.

Thermal behavior of bone and synthetic hydroxyapatites submitted to magnesium interaction in aqueous medium.

The thermal behavior of the products obtained from magnesium interaction with powdered femoral bone and carbonate containing synthetic hydroxyapatite under conditions of pH fluctuation in aqueous medium has been investigated. The products, heat treated at different temperatures from 100 to 1300 degrees C, have been characterized by infrared spectroscopy and X-ray diffraction technique. The results show that the interaction with magnesium ion destabilizes the apatitic structure and favours its thermal conversion into beta-tricalcium phosphate (beta-TCP). The replacement of magnesium with calcium in the beta-TCP crystal lattice hinders its subsequent thermal conversion into the alpha form. The influence of magnesium on the thermal stability is much more evident for carbonate-containing synthetic hydroxyapatite than for bone apatite.