The reduced sensitivity of the ProC Global test in protein S deficient subjects reflects a reduction in the associated thrombotic risk.

To investigate simultaneously a defect affecting the protein C/protein S (PC/PS) anticoagulant pathway is possible thanks to a methodological approach (ProC(R) Global; Dade Behring) based on the activation of endogenous plasma PC by a snake venom extract. Factor V (FV) Leiden, the most frequent cause of hereditary thrombosis, is well detected by the test with sensitivity of 100% irrespective of the presence/absence of thrombosis in the subjects investigated. The test is also suited to detect PC or PS defect, but in this case the in vitro impairment of the PC/PS pathway is less pronounced particularly for PS defects (sensitivity for PC and PS defect, 85-100 and 30-90%, respectively). In this study, we hypothesized that the lower sensitivity described for PS defect, compared with those of PC and FV Leiden defects, could also be related to the clinical condition of the subject investigated (symptomatic/asymptomatic) rather than solely to the PS plasma activity/level. Therefore, we analyzed 126 subjects with single congenital defects in the PC/PS pathway: 46 subjects with PS deficiency (26 thrombotic cases and 20 asymptomatic relatives), 40 subjects with PC deficiency (25 thrombotic cases and 15 asymptomatic relatives), and 40 heterozygous FV Leiden subjects (25 thrombotic cases and 15 asymptomatic relatives). By a cut-off of normalized Agkistrodon contortix snake venom ratio of 0.84, the sensitivity in the whole group of cases (sensitivity a) was 76.1, 95.0 and 100%, respectively, for PS, PC and FV Leiden defects. The test failed to detect 11 (23.9%) among the 46 PS-deficient subjects, and all these cases except two belonged to the asymptomatic subgroup (9/20; 45%). Excluding the 20 asymptomatic relatives, the new sensitivity (sensitivity b) for the PS defect was 92.3%. The comparison of the sensitivity in the symptomatic PS cases and in the asymptomatic ones was significantly different (P = 0.010). Among the 40 PC-deficient subjects, only two (5.0%) were not detected by the test and they belonged indifferently to the two subgroups. Finally, none of the 40 FV Leiden heterozygotes were misdiagnosed by the test. These results suggest that in symptomatic PS-deficient cases the test could reflect a post-thrombotic effect and/or reveal potential unidentified prothrombotic influences assessing a prothrombotic risk condition.

Coexistence of factor V G1691A and factor II G20210A gene mutations in a thrombotic family is associated with recurrence and early onset of venous thrombosis.

Two G-to-A mutations at positions 1691 of the factor V (FV) gene and 20210 of the prothrombin (FII) gene have been associated with an increased risk of venous thromboembolism. We report a thrombosis-prone family in which one subject--the propositus who exhibited combined heterozygous FV G1691A and FII G20210A mutations--showed spontaneous and early clinical onset (at 23 years), recurrences of deep-vein thrombosis and pulmonary embolism. His asymptomatic father carried the FII G20210A substitution and his mother, characterized by an isolated thrombotic episode on occasion of surgery (at 48 years), carried the FV G1691A substitution. In the maternal lineage, one of the propositus' uncles had thrombosis on occasion of a bone fracture (at 65 years) despite the absence of known prothrombotic defects. A sister of the propositus carried the FII G20210A and the brother the FV G1691A mutation. They have been asymptomatic until now. The propositus' two children, 20 and 16 years old, both carry the FV G1691A substitution and have been asymptomatic until now. The plasma levels of FII were higher in carriers of the FII G20210A allele if compared with noncarriers, and the activated protein C resistance phenotype, associated with the FV Leiden mutation, showed a complete correlation with the FV G1691A mutation. Despite the very limited number of thrombotic cases involved in this survey, which does not allow statistically sound conclusions, the data obtained from this family suggest that the synergy of inherited factors and transient risk conditions could play a key role in the occurrence of thrombotic accidents.

A common mutation in the gene for coagulation factor XIII-A (VAL34Leu): a risk factor for primary intracerebral hemorrhage is protective against atherothrombotic diseases.

The role of a common polymorphism in the factor XIII A-subunit gene (FXIII Val34Leu) has been recently investigated as a protective genetic factor against arterial and venous thrombosis. In addition, the less frequent Leu34 allele has been described as a risk factor for intracerebral hemorrhage. We evaluated the prevalence of this polymorphism by PCR in three case-control studies of patients diagnosed as having primary intracerebral hemorrhage (PCH, n = 130), coronary heart diseases (CHD, n = 240; myocardial infarction/no myocardial infarction, 120/120), and cerebrovascular diseases (CVD, n = 240; cerebral infarction/transient ischaemic attack, 120/120). The matched control groups consisted of patients admitted to the hospital without history of vascular disease. In addition, 200 healthy subjects were investigated. The frequency of the mutated allele (Leu34) was higher in patients with PCH than in controls (33.8% vs. 23.1%, P = 0.009) and lower in CHD and CVD patients compared to controls (18.1% vs. 25.2%, P = 0.010 and 17.3% vs. 24.2%, P = 0.011, respectively). Moreover, among the patients with CHD, the Leu34 allele was underrepresented in cases with myocardial infarction than without (12.9% vs. 23.3%, P = 0.004) and than in controls (12.9% vs. 25.2%, P < 0.001). Similar findings were obtained in patients with CVD comparing the cases with cerebral infarction versus cases with transient ischaemic attack (12.5% vs. 22.1%, P = 0.008) and versus controls (12.5% vs. 24.2%, P < 0.001). Finally, considering altogether the groups of ischaemic patients (CHD and CVD, n = 480), it was noted a trend towards a higher mean age of the clinical onset in homozygotes for the Leu allele than in the wild types (P = 0.078). This study indicates that in our population possession of the FXIII Val34Leu mutation predisposes to the occurrence of primary intracerebral hemorrhage and protects against cerebral and myocardial infarction. A wider modulatory role in the progression and onset of atherothrombotic diseases could be ascribed to FXIII Val34Leu.

C677T substitution in the methylenetetrahydrofolate reductase gene as a risk factor for venous thrombosis and arterial disease in selected patients.

BACKGROUND AND OBJECTIVE: Hyperhomocysteinemia, due to a combination of genetic and environmental factors, is considered to be a risk factor for vascular disease. Individuals with the thermolabile variant of methylenetetrahydrofolate reductase (MTHFR), due to homozygous C677T MTHFR gene mutation, have significantly raised plasma levels of homocysteine and may be at increased risk of vascular disease. However, it is still controversial a direct association between C677T homozygosity and the occurrence of vascular disease is still controversial. DESIGN AND METHODS: To clarify the contribution of C677T MTHFR mutation in arterial occlusive disease (AOD) or venous thromboembolism (VTE), we performed a case-controlled study including 160 cases with AOD and 180 cases with VTE attending our referral center and compared them with 200 matched healthy controls. MTHFR gene mutation was evaluated by PCR and odds ratios (OR) and the 95% confidence intervals (CI) were used to estimate the risk for venous or arterial thrombosis. RESULTS: There was a high prevalence of homozygotes for the mutated MTHFR allele among the whole group of cases with arterial disease (OR = 2.35, p = 0.001). Considering the AOD cases with and those without associated risk factors for arterial disease separately the difference remained significant only in the latter group (p = 0.168 and P<0.001 respectively). In contrast, the prevalence of mutated homozygotes among the whole group of cases with VTE was not significantly different from that in the control group (OR = 1.67; p = 0.070). Excluding VTE cases with inherited thrombophilia or with circumstantial risk situations the value increased in both subgroups (OR = 2.26; p = 0.006 and OR = 2.03; p = 0.033 respectively). Considering only VTE cases with neither inherited thrombophilia nor circumstantial risk situations the risk increased further (OR = 2.57; p = 0.017). INTERPRETATION AND CONCLUSIONS: These data suggest that in selected patients homozygosity for the MTHFR mutation increases the risk of both arterial and venous thromboses and that differences in selection criteria for the patient group may be responsible in part for the controversial association of the MTHFR mutation and vascular disease.

Low folate levels and thermolabile methylenetetrahydrofolate reductase as primary determinant of mild hyperhomocystinemia in normal and thromboembolic subjects.

Several studies have indicated that mild to moderate hyperhomocystinemia is a common cause of arterial occlusive disease. Whether hyperhomocystinemia per se is an independent risk factor for vein thromboembolism (VTE) is still somewhat controversial. Both genetic and nutritional factors influence plasma homocysteine levels. Therefore, we evaluated plasma total homocysteine (tHcy), folate, and vitamin B12 levels and established, by polymerase chain reaction, the presence of the C677T mutation (A223V) in the methylenetetrahydrofolate reductase (MTHFR) gene in 220 cases with VTE without well-established prothrombotic defects. As a control group, 220 healthy subjects from the same geographic area as the cases were investigated. Hyperhomocystinemia was defined as a plasma tHcy level above the 95th percentile in the controls (18.05 micromol/L). Hyperhomocystinemia was found in 16% of cases (odds ratio=3.59; P<0.001); deficiencies of folate (<2.47 ng/mL) or vitamin B12 (<165 pg/mL), defined as values below the 5th percentile in controls, were found in 17.7% (P<0.001) and 12.3% (P=0.015) of cases, respectively. The homozygous condition for the MTHFR mutation (VV) was present in 28.2% of cases and 17.7% of controls (odds ratio=1.82; P=0.013). Comparing only the idiopathic forms of VTE (n=80/220; 36.3%) with normal controls, individuals with hyperhomocystinemia, or individuals homozygous for MTHFR mutation increased the odds ratios to 4.03 (P=0.005) and 2.11 (P=0.018), respectively. No statistically significant difference was observed in the MTHFR genotype distribution of cases and controls with hyperhomocystinemia (P=0.386); however, the normal MTHFR genotype (AA) appeared in control subjects only when tHcy levels were below the 80th percentile (10.57 micromol/L) of the distribution, whereas in case patients, it was present at the highest tHcy levels. A strong association between mutated homozygosity (VV), low folate levels, and hyperhomocystinemia was found in both groups. We conclude that in patients with VTE who do not have coexisting prothrombotic defects, hyperhomocystinemia increases the risk of developing idiopathic and venous thrombosis; the homozygous condition for the MTHFR mutation confers a moderate risk but, together with low folate levels, it is the main determinant of mild hyperhomocystinemia in normal and thromboembolic populations.

A modified functional global test to measure protein C, protein S activities and the activated protein C-resistance phenotype.

Identifying a defect affecting the protein C/protein S (PC/PS) anticoagulant system, using a single global test, has recently become possible thanks to a new methodological approach based on the activation of endogenous plasma PC by Protac, derived from Agkistrodon Contortix snake venom (ACV). The introduction of a commercial test (ProC Global), ACV-based, provides a useful tool for the screening of thrombotic patients since the most frequent causes of inherited thrombophilia are found in the PC/PS system. The test provides information only on the global activity of the anticoagulant pathway but not on PC and PS activity or on the factor V related conditions (e.g., FV Leiden). The present study shows that by carrying out the test alternating the presence of PC-, PS-, or FV-deficient plasma and using appropriate amounts of ACV, it is possible to increase the specificity of the test to correctly evaluate respectively the PC or PS activities or the activated protein C resistance condition (APC-R). These simple modifications applied to the original commercial test allow to detect exactly, using a single, basic methodology, the principal defects affecting the PC/PS anticoagulant pathway. Furthermore, carrying out the tests on an automated coagulometer, in combination or not with the classic ProC Global assay, it is possible to use a unique reagent profile to simultaneously investigate in the same or different samples, the PC, PS, and APC-R defect.

Coexistence of antithrombin deficiency, factor V Leiden and hyperhomocysteinemia in a thrombotic family.

We report a thrombotic family with combined type I antithrombin deficiency and factor V Leiden (factor V-R506Q) in which the proposita, affected by recurrent venous and arterial thrombosis, was also characterized by mild hyperhomocysteinemia (28 micromol/l; normal <18.5 micromol/l). Her two thrombotic sisters, with normal antithrombin levels and factor V molecules, showed hyperhomocysteinemia (51 and 30 micromol/l, respectively). Four other members of the family had the combined antithrombin/factor V Leiden defect and two of them had thrombosis. The common A223V mutation in the methylenetetrahydrofolate reductase gene, responsible for the thermolabile variant of the enzyme, was found to be heterozygous in the proposita; the two sisters were homozygous and heterozygous, respectively. The heterozygous sister also had a high titre of antiphospholipid antibodies (85 units of immunoglobulin G antiphospholipid antibody/ml). Furthermore, low plasma folate levels were found in the three hyperhomocysteinemic subjects of the family. This family with several prothrombotic defects is a clear example of the polyfactorial nature of thrombophilia.

Thrombotic risk in thalassemic patients.

Hemostatic parameters of 495 beta-thalassemic patients (421 with thalassemia major and 74 with thalassemia intermedia) were analyzed, to assess their association with the described thrombophilic condition and to verify the role of additional risk factors (e.g. persistent postsplenectomy thrombocytosis, insulin dependent diabetes mellitus, estrogen-progestin treatment and atrial fibrillation). The prevalence of thromboembolic accidents was 5.2% and in four patients (15.3%) inherited or acquired predisposing defects were recognized. The incidence of thromboembolic events and the associated relative risk due to hemocoagulative abnormalities in these patients are discussed.

[Bone marrow scintigraphy in multiple myeloma. A comparison with bone scintigraphy and skeletal radiology]. / La scintigrafia midollare nel mieloma multiplo. Confronto con la scintigrafia ossea e la radiologia scheletrica.

The radiographic patterns of the skeleton of 73 patients affected by multiple myeloma (MM) were compared to the correspondent scintigraphic findings. Whole body scans were performed using 99m Tc-diphosphonates (bone scintigraphy), and 99m Tc-microcolloids (bone marrow scintigraphy). The results indicate that: a) radiography is more sensitive and accurate than scintigraphy in detecting typical myeloma-related bone lesions; b) bone scintigraphy is useful in detecting alterations in particular locations--i.e., sternum, ribs, scapulae, etc.--which are difficult to demonstrate by plain X-rays; moreover, the recovery of the fractures can be visualized; c) bone marrow scintigraphy is employed to demonstrate the presence of marrow expansion, of cold/hot spots, and relative marrow uptake, related to phagocytic activity. Since in adult men red marrow is confined to the epiphysis of long bones and to the spine, all the diseases affecting bone marrow cause medullary expansion/reduction, which are both easily detected by specific radiopharmaceuticals. The peripheral expansion is clearly documented especially in distal humeri and femora since marrow uptake is included, in healthy adults, in the axial and proximal appendicular skeleton. In spite of its yielding unique information, bone marrow scintigraphy remains an additional technique of bone scan, because of its low diagnostic accuracy.

[Value and limits of echography and abdominal lymphography in malignant lymphomas]. / Valori e limiti dell'ecografia e della linfografia addominale nei linfomi maligni.

Diagnostic results obtained by echography and lymphography in the study of abdominal lymphoglandular involvement in patients with lymphomas are compared. The data indicate that while lymphography is more revealing, the two examinations should be used together in staging. Echography, on the other hand, should be used for long-term surveillance, once the lymphographic contrast medium has been absorbed.

Nuclear projections in tumour cells and large chromosome markers.

The application of banding techniques on cytological smears from pleural effusion in a case of histiocytic sarcoma has provided direct evidence for correspondence between nuclear projections in tumour cells and extra large chromosome markers observed in the neoplastic karyotype obtained by direct preparations.

Consecutive cytochemical staining for the analysis of the blastic population in the acute phase of chronic myeloid leukemia.

Blastic crisis in chronic myeloid leukemia is characterized by several cytological alterations which may represent some abortive attempts to differentiate along various cell lines as a consequence of a maturation defect of the myelopoietic cells. These changes of the hematological picture are associated with alterations of the karyotype and with cytochemical abnormalities of the blast cells, possibly related to their metabolic anomalies. In this regard 14 patients with blastic crisis were investigated to achieve an evaluation of the composition of the cell population during the acute phase. A sequence of three cytochemical reactions applied consecutively on the same slide (alpha-naphthyl-acetate esterase + AS D-chloro-acetate esterase + PAS) proved to be useful for the detection of differently oriented blast cells. During the acute phase of chronic myeloid leukemia only about one half of the blast cells were expressing granuloblastic differentiation. The data may be important for some clinical and prognostic factors, since the heterogeneity of the blastic population may be associated with a particular resistance to therapy.