Autologous stem cell transplantation: sequential production of hematopoietic cytokines underlying granulocyte recovery.

We investigated the serum concentrations of a variety of cytokines [granulocyte-macrophage-colony-stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), interleukin (IL) 1 alpha, IL-3, IL-6, IL-8, erythropoietin, tumor necrosis factor alpha, gamma-interferon in 10 patients with advanced ovarian cancer undergoing autologous peripheral blood stem cell (PBSC) harvesting followed by treatment with high-dose cisplatin, etoposide, and carboplatin and PBSC transplantation (chemotherapy was administered on days 1 through 3, PBSCT on day 6). Preliminary observations on cytokine serum levels were performed for 4 patients; on this basis, the kinetics of cytokines was then investigated in greater detail at closely sequential times in 6 further patients. We observed a consistent pattern of sequential GM-CSF, G-CSF, and IL-8 release after chemotherapy/PBSCT in all 10 cases, including the 6 patients monitored in detail: (a) at days 5-10 a GM-CSF peak; (b) at days 12-14 a pronounced release of both G-CSF and IL-8, which always preceded granulocyte recovery by approximately 7 days. At days 17-23, a second GM-CSF peak was monitored in 5 of the 6 patients analyzed in detail, as well as in the other 4 cases. Particularly relevant are the observations that: (a) the peak of G-CSF serum concentration and neutrophil number in the recovery phase are strikingly and directly correlated, thus indicating a key role for G-CSF in granulocyte rescue; (b) the time courses of G-CSF and IL-8 levels are strictly parallel, thereby suggesting a coordinate stimulus for production of granulocytes, mediated by G-CSF, and their activation/migration capacity, mediated by IL-8. Results were essentially negative for IL-3, tumor necrosis factor alpha, and gamma-interferon concentrations (except in one case for each cytokine). An early peak of IL-1 alpha was observed in all 3 analyzed patients, while an IL-6 peak was monitored at days 13-15 in all 4 patients analyzed in detail. The present results indicate a sequential coordinate pattern of cytokine release after ablative therapy and PBSCT and shed light on the mechanisms mediating the recovery of granulocytes, and more generally of hematopoiesis, after stem cell transplantation. Furthermore, these studies may contribute to the design of improved protocols for cytokine administration following myelosuppressive anticancer therapy, as well as to the prediction of granulocytic response.

Antithrombin III molecular variants with defective binding to heparin or to serine proteases: evidence of two different abnormal patterns identified by crossed immunoelectrofocusing.

Molecular heterogeneity of antithrombin III (AT III) was investigated by a technique of crossed immunoelectrofocusing (CIEF) in plasma samples of patients from 16 families with AT III congenital defect, including 8 AT III molecular variants. The AT III CIEF pattern was normal in all the patients with AT III quantitative deficiency, showing a balanced decrease of all the peaks. Out of the 8 AT III variants investigated, 6 had an abnormal pattern: the three variants with defective binding to heparin (AT III Roma, AT III Barcelona, AT III Malmö) shared a similar abnormal pattern; three variants with defective binding to serine proteases (AT III Pescara, AT III Milano, AT III Tampere) had a common abnormal pattern clearly different from the first one, whereas the other two variants deficient in the inactivation of the serine proteases (AT III Chicago, AT III Milano 2) showed a normal pattern. The first type of pathological pattern (type Roma) was characterized by the presence of an abnormal peak overlapping the normal isoforms present at pH 4.8-4.6 and by an additional peak at pH 4.5. The second type of pattern (type Pescara) showed an additional peak at pH 4.5 and an abnormal quantitative distribution of the isoantithrombins all throughout the pH range (5.2-4.6). In order to separate the abnormal AT III fraction from the normal one, plasma of a patient with Roma defect and serum of a patient with Pescara defect were passed throughout an heparin-ultrogel column.(ABSTRACT TRUNCATED AT 250 WORDS)

Hematological causes of venous thrombosis in young people: high incidence of myeloproliferative disorder as underlying disease in patients with splanchnic venous thrombosis.

Thrombotic events occur frequently in myeloproliferative disorders, namely polycythaemia vera and essential thrombocythaemia. Standard diagnostic criteria are designed quite stringent, so that a number of patients could be underdiagnosed. Spontaneous erythroid colonies formation from bone marrow or peripheral blood in the absence of exogenous erythropoietin is considered a reliable index of myeloproliferative disorder even at early stages. Endogenous erythroid colonies (EECs) formation was assessed in 43 patients having recently suffered from venous thrombosis prior to 45 years and without a previous diagnosis of hematological disease favouring thrombosis. A screening for coagulative abnormalities associated with thrombophilia was also carried out: in 5 patients (11.6%) a plasmatic thrombogenic defect was found (quantitative deficiency of antithrombin III, 1 case, protein C, 2 cases, protein S, 1 case, and plasminogen, 1 case). In 10 patients (2 males and 8 females) (23.2%) EECs assay was positive, allowing diagnosis of myeloproliferative disease even though 7 of them did not fulfill standard diagnostic criteria. In the other 3 patients who met the criteria for diagnosis of overt myeloproliferative disease the thrombotic event was the inaugural manifestation. In all these EECs-positive patients thrombosis involved mesenteric and portal veins (n = 4), hepatic veins (n = 3), portal vein (n = 2), mesenteric vein (n = 1). One of them was simultaneously affected from congenital protein C deficiency. Thus latent or atypical forms of myeloproliferative disease as well as the overt stages were the most frequent recognized cause of splanchnic venous thrombosis, accounting for 55% of the cases of our series. On the contrary no EECs-positive subject was found among the 25 patients with other sites of thrombosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Replacement therapy with a purified protein C concentrate during initiation of oral anticoagulation in severe protein C congenital deficiency.

The case of an adult patient with moderately severe protein C deficiency (antigen 16%, activity 12%) is reported. Both parents had protein C levels compatible with heterozygous deficiency. Unlike other reported cases of severe protein C deficiency in adults, the onset of thrombotic symptoms occurred at 1 month of age; however, a symptom-free period until age 17 followed. Replacement therapy with a monoclonal antibody purified protein C concentrate was carried out during the initiation of oral anticoagulation after a course of i.v. heparin for deep vein thrombosis. The administration of the concentrate allowed maintenance of protein C above 50% until a stable therapeutic anticoagulation level could be obtained. This was reached within a short time, thus allowing safe administration of a loading dose of warfarin. We conclude that this approach to the prevention of skin necrosis seems more rapid and safer than previous schedules of oral anticoagulation in protein C-deficient patients.

Instrument effect on the activated protein C resistance plasma assay performed by a commercial kit.

In order to evaluate the influence of the coagulation instrument on the activated protein C (APC)-resistance plasma assay performed by a commercial kit, we tested 70 plasma samples on 4 different instruments during a simultaneous session run using a same lot of Coatest APC-resistance (Chromogenix). The results were analyzed employing three different modes of expression (aPTT prolongation in the presence of APC, APC-sensitivity ratio, normalized APC-sensitivity ratio) and three different diagnostic threshold values (below the control mean--2 standard deviations or the lowest control value or the 5th percentile of the control values). The inter-instruments variability in the mean values of the control individuals can be limited expressing the results as normalized-APC-sensitivity ratio (range 0.99-1.05). The overall diagnostic yield in thrombotic patients and their relatives depended mainly on the instrument employed and only in some cases on the mode of expression of the results and on the diagnostic threshold value. The sensitivity of the commercial assay on heterozygotes for factor V Leiden diagnosed by gene analysis was overall satisfactory (75-100%) but in some cases a lower diagnostic yield was noticed, depending on the type of instrument employed and/or the type of expression of the results and/or the diagnostic threshold values. Thus the instrument system adopted should be carefully considered in the interpretation of the results using the commercial kit.

Management of pregnancy in women with antithrombin III congenital defect: report of four cases.

Four pregnant women with antithrombin III congenital deficiency underwent thrombosis prophylaxis including oral anticoagulants administered from the 16-18th week to the 36-37th week of pregnancy, subcutaneous heparin before the 16-18th week and after the 36-37th week, and a single infusion of AT III concentrate in the peripartum period in order to obtain a minimal level of 0.8 U/ml of AT III functional activity. The level of circulating AT III after the concentrate infusion needs to be evaluated by functional methods, because of a consistent amount in the concentrates of inactive AT III immunoreactive material. No thrombotic or haemorrhagic complication occurred after starting prophylaxis in any woman either in any newborn.

Antithrombin III in full-term and pre-term newborn infants: three cases of neonatal diagnosis of AT III congenital defect.

Antithrombin III (AT III) plasma levels were investigated in 18 full term neonates and 14 healthy preterm neonates. A control group of 20 healthy adults was also studied. AT III was measured as antigen concentration (Ag) and antithrombin or anti-factor Xa heparin cofactor (H.C.) activities. Crossed immunoelectrophoresis on heparin-agarose (H-CIE) was carried out on plasma samples; moreover the distribution of isoantithrombins was investigated on whole plasma by a technique of crossed immunoelectrofocusing (CIEF). AT III plasma levels in full term infants were significantly lower as compared to the adult values. The preterm newborns group showed a further significant decrease in AT III levels as compared to the full term neonates. In all infants AT III H-CIE runs displayed a single fast moving anodal peak, so that a normal binding to heparin was demonstrated. The CIEF AT III plasma pattern of the adults as well as of all neonates displayed three major peaks at pH range 5.2-4.9, a small amount of AT III at pH 4.9-4.8 and a minor peak at pH 4.8-4.6, so that it was concluded that the isoantithrombins plasma distribution in neonatal age is identical to that of the adult subjects. Four neonates whose mothers were affected by AT III congenital defect were also investigated: diagnosis of congenital deficiency was established in three cases.

Clinical manifestations and management of inherited thrombophilia: retrospective analysis and follow-up after diagnosis of 238 patients with congenital deficiency of antithrombin III, protein C, protein S.

The clinical history of 238 patients with inherited thrombophilia (AT III = 94, PC = 103, PS = 41) was analyzed retrospectively at diagnosis and in the follow-up period after diagnosis. At diagnosis 129 patients (54%) had suffered from thrombosis, with a recurrence rate of 48%. The most frequent onset manifestation was deep vein thrombosis of lower limbs (58%). Thrombotic history started before 40 in 80% of the cases. Forty-nine percent of the venous thromboses were preceded by a triggering event, in most cases pregnancy (17%) and surgery (12%). After diagnosis, follow-up lasted a total of 1,113 pt-years. A policy of short-term prophylaxis during risk situations for all patients and long-term prophylaxis in symptomatic patients failed to prevent venous thrombotic episodes (diagnosed by objective methods) in 4 previously asymptomatic subjects and recurrence in 7 previously symptomatic subjects. After knowledge of the patients' diagnosis the incidence of venous thrombosis/100 pt-years was reduced as compared before diagnosis as total episodes (onset+recurrencies) (1.0 vs 1.9), onset episodes (0.7 vs 1.3) and recurrent episodes (1.3 vs 4.8), even though the differences were not statistically significant. However most of the venous thromboses occurred at a more advanced age (67% after 40 years) and without any apparent cause (83%), at significant variance with the period preceding the diagnosis; in particular the incidence of venous thrombotic onset in patients younger than 40 passed from 1.3/100 pt-years to 0.2/100 pt-years. In 6 recurrences after diagnosis a poor compliance for antithrombotic treatment was recognized.(ABSTRACT TRUNCATED AT 250 WORDS)

Prognostic factors of patients with acute myeloid leukemia (AML) allografted in first complete remission: an analysis of the EORTC-GIMEMA AML 8A trial. The European Organization for Research and Treatment of Cancer (EORTC) and the Gruppo Italiano Malattie Ematologiche Maligne dell' Adulto (GIMEMA) Leukemia Cooperative Groups.

The Leukemia Cooperative Groups of the EORTC and the GIMEMA conducted a prospective randomized phase III trial, in order to assess the value of autologous BMT (ABMT) vs a second intensive consolidation course (IC2), following a common intensive consolidation course (IC1) for patients with AML. Patients with an HLA-identical sibling donor were not randomized, but were included in an allogeneic BMT (alloBMT) program. This is an analysis of prognostic factors which influence the outcome of treatment after alloBMT in first complete remission (CR). The study included 730 patients < 46 years of age in CR, 270 having a histocompatible sibling donor. In 169 of these patients alloBMT was performed in first CR. Early remitters (122 patients achieving CR with one course of treatment) had a DFS at 3 years of 67%, significantly longer than that of 44% for late remitters (47 patients achieving CR after more than one course of treatment) (P = 0.006). The relapse risk for early vs late remitters was 16 and 40% at 3 years (P = 0.001) and the treatment-related mortality (TRM) at 2 years was 21 vs 27%. Age appeared to be a prognostic factor for TRM, WBC for DFS, whereas the FAB classification was not of prognostic importance. Patients with poor risk cytogenetic abnormalities showed a trend towards a higher relapse risk. Patients transplanted shortly after achieving CR appeared to have a worse prognosis than those transplanted further into remission. Overall, the number of courses of induction therapy needed to achieve CR was the most important prognostic factor for outcome after allogeneic BMT.

A method for the differential determination of plasma antithrombins.

A method for the differential determination of plasma antithrombins, antithrombin III and alpha2 macroglobulin, is described. The method is based on the selective inactivation of plasma alpha2 macroglobulin by treatment with 0-1 M methylamine for 10 minutes at 37 degrees C and on the observation that antithrombin III and alpha2 macroglobulin inhibited in defibrinated plasma low concentrations of thrombin without mutual interference and according to pseudo-first order reaction. In healthy subjects antithrombin III was shown to account for about 70% of the total antithrombin activity. But in patients with liver cirrhosis, where low levels of total antithrombin activity were observed, the relative contribution of antithrombin III was found to be noticeably lower.

Discrepancies between immunological and caseinolytic plasma plasminogen assays in health and hyperfibrinolytic states.

A sensitive immunological assay for plasminogen and/or plasmin was developed by using a haemagglutination inhibition technique. Plasma and the corresponding euglobulin fraction of healthy subjects and of patients with hyperfibrinolysis (liver cirrhosis, thrombolytic treatment) were assayed for plasminogen using immunological and caseinolytic techniques. In hyperfibrinolytic states discrepancies were found between immunoreactive and caseinolytic plasma plasminogen whereas a good correlation was observed between immunoreactive and caseinolytic euglobulin plasminogen. Experiments in vitro suggested that these discrepancies might depend on the presence in hyperfibrinolytic plasma of variable amounts of plasmin(ogen)-related antigen which has no caseinolytic activity and is not precipitated with euglobulin.

Acute Nonlymphoblastic Leukemia: Treatment of Elderly Patients.

Forty-six elderly patients with acute non lymphoblastic leukemia (ANLL) were treated with a low toxicity drug combination (Oncovin, low dose Ara-C and Prednisone (OAP)) or with much more aggressive regimens. Complete remission was achieved in 6/23 patients treated with OAP and in 9/23 with aggressive chemotherapy (AC). The mean duration of remission was 18 and 27 weeks, respectively. Ten patients in the first group and 5 patients in the second group were resistant to therapy. During OAP treatment, 7 patients died, 6 during the induction phase and 1 in the consolidation phase, while 9 patients in the group treated aggressively died during the induction phase We conclude that aggressive regimens may be used in well selected elderly patients while patients with severe preexisting medical diseases may be treated with less aggressive drug combination.

Further characterization of a pathological isoantithrombin with no affinity for heparin (antithrombin III Roma).

A molecular antithrombin III variant (Antithrombin III Roma) with an abnormal pattern of crossed immunoelectrofocusing was further investigated in order to identify the pathological isoforms. AT III crossed immunoelectrofocusing of the whole plasma from the affected patients showed a loop overlapping the peak normally present at pH 4.8-4.6. Affinity chromatography demonstrated the presence of an AT III fraction totally lacking in heparin affinity. Crossed immunoelectrophoresis on heparin-agarose (H-CIE) and crossed immunoelectrofocusing (CIEF) runs performed on the fractions obtained by heparin-agarose affinity chromatography confirmed that the functional defect was exclusively related to the pathological isoantithrombin (pH 4.8-4.6), which was also devoid of any progressive activity. The AT III fraction with normal affinity to heparin displayed H-CIE and CIEF patterns identical to the control AT III.

Mesenteric vein thrombosis in protein S congenital deficiency.

Mesenteric vein thrombosis is considered an uncommon clinical presentation of protein S congenital deficiency. In the two patients with mesenteric vein thrombosis here reported an isolated deficiency of protein S was diagnosed; family investigation recognized protein S deficiency also in five relatives of one of them.

Association of Graves' disease and prekallikrein congenital deficiency in a patient belonging to the first CRM+ prekallikrein-deficient Italian family.

Severe prekallikrein (Fletcher factor) deficiency was diagnosed in a 49-year-old woman and in 3 of her siblings. Functional prekallikrein (PKK) activity was found below 1% by clotting assay and 20% by amidolytic assay in all the affected subjects; PKK cross-reacting material (CRM) was present in all the patients (antigen levels from 34% to 54%). This is the first CRM+ PKK-deficient family identified in Italy. The index patient was affected from Graves' disease: such association was previously reported in another patient with PKK congenital defect.

Factor VIII complex in progressive systemic sclerosis.

Factor VIII complex and its related activities (Coagulant, Antigen and Ristocetin Cofactor) have been investigated in 23 patients with Progressive Systemic Sclerosis (PSS) divided into two groups: acrosclerosis and diffuse sclerosis. All Factor VIII-related activities were higher in PSS patients than in normal subjects. No difference in F. VIII-related Antigen (F. VIIIR:Ag), F. VIII-related Ristocetin Cofactor (F. VIIIR:Co) and F. VIII Coagulant activity (F. VIII:C) was found comparing the patient groups. F. VIII:C was increased significantly less than F. VIIIR:Ag and F. VIIIR:Co in both patient groups. Some hypotheses about the pathogenesis of this increase are discussed.

Serum lactate dehydrogenase isoenzyme pattern in non-Hodgkin's lymphomas.

Serum lactate dehydrogenase (S-LDH) and its isoenzyme pattern were assayed in 63 non-Hodgkin's lymphoma (NHL) patients, 37 at diagnosis, 15 at relapse and 11 in complete remission (CR). S-LDH in NHL patients with active disease was higher than in normal subjects and CR patients (p less than 0.001). Among the isoenzymes, LDH-2 and LDH-5 showed no remarked differences; LDH-1 was reduced and LDH-3 and LDH-4 raised in comparison to the normal group (p less than 0.001). S-LDH levels and isoenzymes 1 and 4 were influenced by the stage, the histological subgroup and by the presence of general symptoms. In fact, cases in stage IV, with "high-grade malignancy" and with general symptoms, had higher S-LDH levels and more evident LDH-1 and LDH-4 changes than the other stages, the other histopathological subgroups and the cases classified as "A". S-LDH was the same as in normal subjects in the "low-grade" and "intermediate-grade" malignancies as was LDH-1 in stage II and LDH-4 in stages II and III, in "low-grade" malignancy and in the A cases. In contrast, LDH-3 was always high, with no significant difference in relation to the variables considered. Thus, in NHL, LDH-3 seems to be a reliable marker of the presence of the disease in any case, whereas S-LDH is more related to the spread of the lymphoma.

Urokinase and AT-III concentrate treatment in inferior vena cava thrombosis associated with nephrotic syndrome.

A patient with an inferior vena cava thrombosis in nephrotic syndrome was treated with heparin, AT-III concentrates and urokinase. After a few days on treatment he showed a complete resolution of the thrombosis. We suggest that this therapeutic combination may be a good approach to the treatment of thrombosis in nephrotic syndrome.

Cystic fibrosis complicated by Hodgkin's lymphoma.

The case of a patient with cystic fibrosis who developed an Hodgkin's lymphoma is reported. ABVD chemotherapy induced complete remission but caused a worsening of pulmonary function.

Serum beta 2-microglobulin in systemic sclerosis.

The role of beta 2 microglobulin (beta 2-m) in Systemic Sclerosis (SS) has been evaluated. Twenty-four female patients have been examined: 15 of them were affected by acrosclerosis (Group 1) and 9 of them by diffuse sclerosis (Group 2). 46.6% of Group 1 and 44.4% of Group 2 had values significantly higher than normal controls. (P less than 0.01 and P less than 0.005 respectively). The authors deal with the validity of the use of B2-m as index of inflammatory activity of the disease.