B. Bizzi

Arterial thrombosis as clinical manifestation of congenital protein C deficiency

SummaryA 49-year-old man was hospitalized for slight paresis of the upper left limb. Thrombosis of the right internal carotid artery was documented by arteriography and digital angiography, which showed evidence of an anastomotic blood flow. He went on anticoagulation treatment. Five years later, after an uneventful period, he was referred to our center for the occurrence of a superficial thrombophlebitis: diagnosis of congenital protein C deficiency was possible in the patient as well as in two of his relatives. Two other subjects with congenital protein C deficiency belonging to two different kindreds, whose illness was diagnosed in our center, suffered from myocardial infarction and TIA, respectively, as the only clinical manifestation; a fourth case, previously described, with recurrent superficial thrombophlebitis, suffered from a TIA when on treatment with stanazolol. These cases indicate that arterial thrombosis or TIA is not an uncommon event in congenital protein C deficiency, even in patients without other risk factors for arterial thrombosis.

Antithrombin III Pescara: a defective AT III variant with no alterations of plasma crossed immunoelectrophoresis, but with an abnormal crossed immunoelectrofocusing pattern

Summary. An abnormal AT III variant was found in five members from a family where a high incidence of thromboembolism occurred. In all the affected subjects AT III antigen concentration was normal, whereas antithrombin and anti‐factor Xa progressive activities as well as heparin cofactor activities were low. Crossed immunoelectrophoresis performed either in absence or in presence of heparin showed a normal plasma pattern. Further chromatographic investigations showed a normal affinity to heparin. An abnormal plasma pattern was evidentiated by crossed immunoelectrofocusing throughout all the AT III pH range. These data are consistent with the presence of an abnormal AT III variant with a defective binding to serine proteases and clearly identifiable only by crossed immunoelectrofocusing. This variant appeared different from the other qualitative AT III defects so far described and was named ‘Antithrombin III Pescara’.

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.

Identification of blast cells in peripheral blood through automatic assessment of nuclear density: a new tool for monitoring patients with acute leukaemia

The Technicon H*1 haematology system is provided with a new method for basophil count, neutrophil lobularity assessment and detection of blasts in peripheral blood through automated measurement of nuclear density. We compared the results of the H* 1 blast flag with those of the microscope examination in 131 peripheral blood samples from 43 patients with acute leukaemia in different phases of their disease, to determine the degree of sensitivity and specificity of the system in this setting. In six patients at diagnosis or in overt relapse, all having large percentages of blasts at the manual differential count, a typical deformation of the profile of the mononuclear cell population on the display was consistently observed, regardless of the morphological subtype of leukaemic cell which was involved. Amongst 34 samples with 4–95% morphologically recognizable blasts on the peripheral blood film, the sensitivity of the system was 100%. with no false negatives at all. In 43 samples, on the other hand, the H*1 blast flag was positive in the absence of any morphological evidence of blasts on the smear. These ‘false positives’, however, were always obtained from leucopenic patients who had more than 12% blast infiltration in the bone marrow, compared to the 0–6% value that was found in patients with a negative H*1 blast flag. These results suggest that the H*1 system is a highly sensitive tool for the detection in peripheral blood of even small concentration of leukaemic cells, which escape morphological identification.

Antithrombin III during High-Dose Cytosine Arabinoside Therapy with or without Asparaginase

Fourteen patients with hematologic neoplasia (11 acute myeloid leukemias, 2 non-Hodgkins lymphomas and 1 blast crisis of chronic myeloid leukemia) who underwent high-dose cytosine arabinoside (HIDARAC) therapy with or without sequential asparaginase (ASNase) were investigated in order to evaluate liver toxicity and a possible decrease in antithrombin III (AT III) plasma level. AT III was found decreased only in patients who received ASNase, whereas HIDARAC alone did not influence AT III levels. It is pointed out that a single dose of ASNase seems to be sufficient to induce a decrease in AT III. A mild and transient liver toxicity due to HIDARAC therapy does not seem to be of any clinical relevance.

Tamoxifen-induced immune-mediated platelet destruction. A case report.

Drug-induced immunologic thrombocytopenia, a fairly common disorder, is characterized by drug-dependent antiplatelet antibodies that destroy circulating platelets in the presence of the provoking drug or its metabolites. The development of reliable methods for the detection of platelet-bound immunoglobulins causing in vivo platelet destruction, such as the use of monoclonal antibodies tagged with fluorescein and flow cytofluorimetric analysis, has ushered in a new era to differentiate between immune and non-immune thrombocytopenias. A severe thrombocytopenia developed in an elderly female patient treated with tamoxifen, a non-steroidal antiestrogen drug, after surgery for breast cancer. A tamoxifen-dependent platelet antibody was detected in the patients serum and linked on the platelet membranes. This antibody reacted only in the presence of the offending drug and showed platelet specificity. Withdrawal of drug restored platelet count to normal levels.

Effects of a preformed extracellular matrix on long-term serum-free bone marrow culture.

SummaryThe extracellular matrix (ECM) produced by the stromal layer plays a key role in the regulation of commitment and differentiation of hematopoietic cells. Long-term bone marrow culture (LTBMC) allows analysis of the stromal microenvironment. Recently, serum-free LTBMC has been described, but the formation of a classical adherent layer was never observed under these conditions. We have evaluated the effect(s) of a chemically well defined ECM on serum-free and serum-dependent LTBMC. In serum-dependent cultures ECM did not induce a significant increase of hematopoiesis. In serum-free conditions, a marked improvement of hematopoiesis was observed, both in terms of CFU-GM and BFU-E yield and in duration of cultures. A confluent stromal layer was observed only in the presence of ECM. The present results indicate that the addition of ECM to serum-free cultures provides a standardized culture condition, while improving progenitor cell recovery and allowing formation of a confluent stromal layer. Moreover, ECM+ LTBMC may provide a model to study the effect(s) of adhesive proteins and hematopoietic growth factors normally present in serum.

ASSOCIATION OF CONGENITAL PROTEIN C DEFICIENCY AND LATENT MYELOPROLIFERATIVE DISEASE AS CAUSE OF SPLANCHNIC VENOUS THROMBOSIS IN A 34‐YEAR‐OLD WOMAN

Protein C is a vitamin K-dependent glycoprotein with anticoagulant activity. Hereditary deficiency of protein C is heterogeneous in clinical expression: family studies showed kindreds with a high incidence of thrombosis as well as kindreds where the defect was symptomatic only in the homozygotes or in a very few of the heterozygous subjects (Pabinger. 1986). A large number of asymptomatic heterozygotes was identified by an extensive study on healthy adults (Miletich et al, 1987). It is unclear if this heterogeneity is due to the existence of unrecognized subtypes of the defect or to the presence in some subjects of other factors predisposing them to thrombosis. Here we report a case of splanchnic venous thrombosis in a heterozygous woman belonging to an asymptomatic protein C deficient family and affected by a latent myeloproliferative disorder. A 34-year-old woman whose previous history had been free of thrombosis was admitted to our hospital with severe abdominal pain. An ultrasound examination showed splenomegaly and evidence of splanchnic venous thrombosis; a celiac angiography showed thrombosis of the splenic, superior mesenteric and portal veins. The patient received heparin i.v. and a local infusion of urokinase followed by oral anticoagulant treatment. On admission, laboratory data showed a haemoglobin level of 12.9 g/dl, red cell count 5.01 x 10L2/1 haematocrit 40.6%. MCV 81 fl, white cell count 19.6 x 109/1, with 81% neutrophils, 9% lymphocytes, 8% monocytes. 1% eosinophils and 1% basophils, platelet count 376 x 109/1, serum iron level 4.5 pmol/l: a bone marrow smear examined cytogenetically was normal. Arterial oxygen saturation was 92.9%. Antibodies to DNA and nuclear antigens were absent; a test for lupus-like anticoagulant was negative. Antithrombin I11 (antigen and heparin cofactor activity), protein S (antigen), plasminogen (antigen and activity) and heparin cofactor I1 (activity) were normal. Protein C antigen (electroimmunoassay) was 46% with factor I1 and X antigens of 9 7% and SS%, respectively: protein C functional activity (snake venom clotting method) was 40%. The family study showed a protein C deficiency in the father and in three children of the propositus, with antigen values ranging from 59% to 70% (normal range 70-140%) and functional activity values ranging from 64% to 68% (normal range 70-140%); the family history was negative for thromboembolic events. The patient was discharged after prescribing oral anticoagulant treatment for life and iron therapy administered orally. During the following 12 months periodic tests showed a progressive increase in platelet count (up to 575 x 109/1) and a slight leucocytosis (around 10 x 1 @/I): serum iron rose to 7.16 pmol/l whereas haemoglobin and red cell values count remained substantially unchanged as compared to the values on admission. The fact that the family history was negative for thrombosis, together with the rare occurrence of mesenteric vein thrombosis as the presenting manifestation of hereditary protein C deficiency, as well as the progressive increase in the platelet count and the slight leucocytosis. prompted us to investigate the presence of a concurrent cause of splanchnic venous thrombosis such as a myeloproliferative disorder. Formation of erythroid colonies (EC) from bone marrow or peripheral blood without addition of exogenous erythropoietin in the culture medium was evaluated according to Cashman et aZ (1 983). with minor modifications. In both tests EC were evident after 10 d (52 colonies/2 x lo5 seeded cells in the bone marrow assay and 93 colonies/2 x lo5 seeded cells in the peripheral blood assay). The test was also positive in two more assays carried out on peripheral blood after 1 month and 2 months, respectively. Benzidine staining of the smears obtained from the colonies after cytocentrifugation showed the presence of haemoglobin in the colony cells (Fig 1). Fourteen months after the acute thrombotic episode the total erythrocyte volume was 31 ml/kg (normal value in women 25 ml/kg): serum BI2 was 719 ng/l (normal range 160-9 70) and the serum erythropoietin level (ELISA) was 18 mU/ml (normal range 22-54). Spontaneous EC formation is considered a reliable sign of polycythaemia Vera or myeloproliferative disorder, showing the presence of an abnormal cellular clone with hypersensitivity to the minimal amounts of erythropoietin present in the culture medium even at early stages of the disease (Lemoine et a/ , 1986). Spontaneous EC formation was shown in 16 of 20 patients with Budd-Chiari syndrome and in 14 of 33 patients with portal vein thrombosis (Valla et a/, 1985, 1988): in this latter series only three subjects who were EC positive had an overt myeloproliferative disease at the time of diagnosis, but a further four developed an overt myeloproliferative disorder between 6 months and 5 years after the acute episode. The case reported here did not fulfil the standard criteria of the Polycythemia Vera Study Group (PVSG) for diagnosis of polycythaemia Vera (Wasserman, 1971). showing only two major criteria (splenomegaly, which could be due also to portal hypertension, and arterial oxygen saturation > 92%) and one minor criterium (thrombocytosis >400 x 10y/l). However although red cell volume did not meet the PVSG criterium (2 32 ml/kg for women), it was increased and the erythropoietin level was decreased. Thus, the formation of spontaneous EC allowed the diagnosis to be made of primary myeloproliferative disorder at an early stage in a subject heterozygous for protein C deficiency. Severe splanchnic

DISAPPEARANCE OF SPONTANEOUS ERYTHROID COLONIES IN PATIENTS WITH MYELOPROLIFERATIVE DISORDERS TREATED BY ALPHA-INTERFERON

Recombinant alpha-interferon has been demonstrated to be effective in reducing platelet count in patients with essential thrombocythaemia (ET) (Giles et al, 1988). More recently the activity of interferon in controlling the red cell mass in patients with polycythaemia Vera (PV) has been also investigated, with encouraging results (Silver, 1990). In both these myeloproliferative diseases the presence of endogenous erythroid colonies (EECs) (either CFU-E or BFU-E) has been demonstrated, as expression of the hypersensitivity to erythropoietin of the neoplastic clone (Eaves & Eaves, 1978; Eridani et al. 1983). In the present study we examined the behaviour of the EECs in five subjects during treatment with alpha-interferon (Intron A. Schering Plough) (Table I). One patient (case 1) had ET and two patients (cases 2-3) had PV. according to the Polycythaemia Vera Study Group (PVSG) criteria (Wasserman, 1971); one patient (case 4) fulfilled all criteria for ET diagnosis, except for the presence of the Philadelphia chromosome, in the absence of leucocytosis, as occasionally observed in a subset of Ph-positive patients (Stoll et al. 1988). In one patient (case 5) with a previous history of portal vein thrombosis the PVSG diagnostic criteria were not met, but EECs were detected either in bone marrow or peripheral blood: bone marrow biopsy showed megakaryocytic dysplasia and fibrosis. Therefore a diagnosis of latent myeloproliferative disorder was suspected, as previously described in a similar series of thrombotic patients (Teofili et al, 1992). In the four patients with overt myeloproliferative disease interferon treatment consisted in S . C . 3 MU daily (cases 3 and 4) or 3 MU every other day (cases 1 and 2). Two of them (cases 1 and 4) were newly diagnosed; two patients were previously treated with phlebotomies (case 3) or phlebotomies and chemotherapy (case 2). In the patient with latent myeloproliferative disorder alpha-interferon was given as unique course of S.C. 3 MU daily for 15 d, after oral informed consent. EECs were evaluated, as previously described (Teofili et al. 1992) in peripheral blood (all cases) and bone marrow (cases 3 and 5) before and after 2-6 weeks of therapy. In three cases the in vitro addition of exogenous alpha-interferon (Intron A, Schering Plough 10, 100, 1000 lJ/ml) was also evaluated. Before starting treatment EECs were present in all patients either in peripheral blood and, in the two cases evaluated, in bone marrow samples. After 2 weeks of therapy EECs disappeared in patient 5 but persisted in patient 3 ; comparable results were obtained in bone marrow and peripheral blood in both cases. After 4 weeks of treatment EECs were absent in all patients evaluated except in case 3 ; after 6 weeks EECs were absent in all patients. Haematological parameters correlated well with the EEC’s behaviour. During interferon administration, peripheral blood BFU-E and CFU-GM showed a progressive decrease in all but one patient (case 3 ) (Table I). Three out of four patients with overt myeloproliferative syndrome are still on treatment with alpha-interferon, with a good control of the disease (cases 1 , 2 and 4, with a follow-up

Effect of Oral Anticoagulant Treatment on Plasma and Serum Antithrombin III: A Study on 172 Patients at Different Levels of Anticoagulation

Antithrombin III (AT III) functional levels are much lower in serum than in plasma; during oral anticoagulation this difference is reduced. Plasma and serum of 172 patients taking vitamin K antagonists were tested for AT III antigen and both AT III heparin cofactor and anti-Xa heparin cofactor. Crossed immunoelectrophoresis of AT III on heparin-agarose was also carried out in plasma and serum. The patients were divided into four groups: (1) international normalized ratio (INR) 9.3-4.1, n = 25; (2) INR 4.0-2.5, n = 73; (3) INR 2.4-2.0, n = 40, and (4) INR 1.9-1.5, n = 34. 66 healthy subjects were used as controls. Plasma levels of AT III antigen, AT III heparin cofactor, and anti-Xa heparin cofactor were the same in all groups. In all groups all serum AT III parameters were higher than in controls; crossed immunoelectrophoresis of AT III on heparin-agarose indicated that this finding was due to a lower formation of complexed AT III in serum. AT III heparin cofactor serum values were the same whatever the INR over a large range (9.3-1.5); the highest anti-Xa heparin cofactor serum levels were noted in the groups treated more intensely (groups 1 and 2).