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A flow cytometric method to detect apoptotic cells is described. This method is based on the detection of differences in chromatin condensation with Hoechst 33342 as a probe and the detection of dead cells with propidium iodide as a probe for membrane damage. By this method it was possible to detect, in the same sample and at the same time, intact cells, cells undergoing apoptosis, and dead cells resulting from apoptotic and/or necrotic processes. The method was successfully applied to the detection of apoptotic cells in two human cell models: cultured polymorphonuclear cells and the U937 cell line treated with antitumoral drugs. Staining specificity for apoptotic cells was controlled by cell sorting of the presumed apoptotic population, followed by morphologic examination or DNA analysis of the sorted populations. The usefulness of such a method is discussed in terms of applications in the analysis of heterogeneous clinical samples, populations with low DNA degradation during apoptosis, and cell cycle position of the apoptotic cells.

Cytarabine (Ara-C) is currently used in the treatment of adult acute myeloid leukemia (AML). To predict the results of induction chemotherapy, it could be useful to detect leukemic cells that are resistant to Ara-C in patients with AML. Using a bromodeoxyuridine/DNA (BrdUrd/DNA) staining method in flow cytometry (FCM), we have developed a cell resistance index to Ara-C (RI). The technique has been applied to 121 bone marrow (BM) samples from patients with de novo AML treated by a regimen containing Ara-C and daunorubicin (DNR). Ninety-seven patients achieved a complete remission (CR), and 24 patients did not and were considered drug-resistant (DR). The BM cells collected at diagnosis were cultured for 48 hours and underwent BrdUrd/DNA analysis. Among 25 patients with no or very low proliferative activity (<3% of cells in S-phase), the proportion of DR patients (nine of 25) was significantly higher than in a second group of 96 patients with detectable proliferative activity (15 of 96) (P < .025). Within this second group, there was a first group of nine patients with high RI values, which included only DR patients; a second group of 63 patients with low RI values, which included 62 CR patients; and a third group of 24 patients with intermediate RI values, which included 19 CR and five DR patients. In view of this series, our results show that it is possible to detect a majority of DR patients treated by Ara-C.

Fatty acid profiles of various lipid fractions were determined in carefully selected insulin-dependent and non-insulin-dependent diabetics to assess relationships between serum fatty acid composition and type of diabetes. Clear-cut hypertriglyceridemia with slight hypercholesterolemia was found in both diabetic types. The decrease of lignoceric acid in sphingomyelin is the only alteration found in both types of diabetes. In the insulin-dependent diabetics, there were increases in levels of oleic acid and of alpha-linolenic acid in esterified cholesterol, and in levels of alpha-linolenic acid in the triglyceride fraction. In the non-insulin-dependent diabetics, there were increases in levels of oleic acid and total monounsaturated fatty acids in the triglyceride fraction and there was an increase in levels of saturated fatty acids and a decrease in levels of polyunsaturated acids in phosphatidylcholine; in sphingomyelin, dihomogamma-linoleic acid levels were enhanced. Arachidonic acid levels were normal in our patient population.

Microvessels, especially in the skin and muscles are organized in functional units. These units are controlled by the adrenergic system and hormones but also have autonomous metabolic and myogenic regulatory systems independent of vasomotion. Microcirculatory blood flow is affected by special rheologic properties: a low arteriolar haematocrit rises to the systemic level in the venules. The flow rate in the venules is low, and together with the raised haematocrit, explains the microvenular sensitivity to hyperviscosity. Capillarovenular microangiopathy, recently described by standard and fluorescent capillaroscopy, develops during chronic venous insufficiency. The capillary loops appear dilated and knotted together with fibrous deposits and pericapillary oedema. Venous hyperpressure is the cause of this microangiopathy. Together these phenomena disrupt normal haemodynamics and physiology of the microcirculatory unit: baseline hyperhaemia, lowered vasomotor and vasomotion reactivity, development of rheologic disorders (haemoconcentration, hyperfibrinogenaemia, erythrocyte agregation) and decreased fibrinolysis. Modifications in the transcapillary exchange is related to hypoxia and is aggravated by depressed lymphatic drainage. The main consequence is oedema. Inflammation, a characteristic of these microangiopathies could occur when the endothelium is activated by the hypoxia. The classical mediators of inflammation would activate interactions between the different cells: endothelium, granulocytes, monocytes and platelets. Several pharmacological models have been developed for the analysis of these data including exploration of the permeability and capillary resistance and rheological analysis. Objective observation of the microangiopathy with capillaroscopy, together with modern haemodynamical, biological and pharmacological methods are essential for a better understanding of microvascular disorders in chronic venous insufficiency.

The antigen Ki-67 Ag, regarded as a marker for proliferating cells, was identified as a protein(s) (pKi-67) which can exist free or associated with DNA as evidenced by DNA digestion of cells before or after immunolabeling with Ki-67. The dual nature of this antigen was also supported by reconstitution of Ki-67 Ag from purified DNA and nuclear proteins extracted from the K562 cell line. The immunoreactivity of the resulting complexes was examined in solution using Ki-67 and MIB-1 antibodies. The interaction between Ki-67 or MIB-1 antibodies and pKi-67 was enhanced in the presence of undegraded ds DNA, indicating that ds DNA modulates the conformation of pKi-67 and that the altered conformation of pKi-67 is more reactive than the pure protein to both Ki-67 and MIB-1 antibodies.

Clinical or pharmacological trials require reproducible laboratory tests. The aim of this work was to investigate the conditions of quality control of two laboratory tests, blood viscosity and red cell aggregation used for hemorheology trials in three centers. The results showed that viscosity measurements require 2 standards. For red cell aggregation, the control suspension standardized media (normal an pathological aggregation) showed a good intercentre reproducibility of the dissociation thresholds but a wide variability of aggregation times. Complementary studies in 5 or 6 centres are planned in order to define national standards of quality control.

Endothelium damage is associated with thrombotic risk in a variety of diseases including atherosclerosis, gram negative sepsis, viral infections and neoplastic disease. Therefore, it appears necessary to find a mean for the clinical investigation for such a damage. Among the markers of these cells, thrombomodulin which is a membrane glycoprotein, seems to be of great interest for this purpose. Actually, thrombomodulin is also found in plasma, following an endothelial lesion. Plasma levels of thrombomodulin are increased in a certain number of pathologies associated with endothelium lesion: atheromatous arterial disease, disseminated intravascular coagulation syndrome and also in systemic lupus erythematosus where the levels of plasma thrombomodulin are related to the severity of the pathology. Moreover, previous in vitro studies confirm the fact that the release of thrombomodulin from the endothelial cell membrane occurs during the course of injury by activated leukocytes or hydrogen peroxide. So, one can suppose a prospective interest in the measurement of plasma thrombomodulin as a diagnostic tool for the approach of endothelium damage.

The plasma thrombomodulin (TM) level depends on the integrity of the endothelium and the clearance of the molecule. In several different pathological conditions, plasma TM levels increase with damage to the endothelium. We studied plasma TM levels in patients with various localizations of atheromatous arterial disease who had normal serum creatinine levels. Two groups of patients had a single symptomatic localization, which was either peripheral occlusive arterial disease (POAD) or ischemic heart disease (IHD) and a third group of patients had multiple symptomatic localizations (polyvascular). We compared the plasma TM levels with the plasma levels of other specific markers of endothelial cell activation such as: prostacyclin (PGI2), tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1). Plasma TM levels were significantly increased in all three individual groups and when all patients were considered (total patients), as compared with normal controls. When all patients were considered, there was a significant positive correlation between plasma TM levels and t-PA and between plasma TM levels and PGI2. A significant positive correlation was also found between the plasma TM levels and PAI-1 for patients with POAD. Thus, our findings suggest that an increased influx of TM into the plasma may be caused by endothelial cell damage in patients with atheromatous arterial disease. However in our study, the plasma TM levels obtained were similar for all three types of atheromatous arterial disease. Though plasma thrombomodulin is a marker of endothelial cell injury, it cannot be of a clinical interest until its levels are related to the extend of the atheromatous lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the quantity and activity of the Protein C molecule is of the utmost importance in highly purified concentrates prepared for replacement therapy. A multicenter study was undertaken to evaluate the comparability and accuracy of Protein C assays from commercial sources. Significant between-assay and interlaboratory differences were found for both functional and immunological assays. The interlaboratory variability is explained in part by the use of different control plasmas. The results also indicate the importance of the diluent used. This study emphasizes the need for standardized methods for determining the characteristics of Protein C concentrates.

The hemorheologic and fibrinolytic variables of 15 patients undergoing elective aortic graft surgery were investigated before, during, and after surgery. During the operation, a relative hemodilution was induced intentionally by an infusion of crystalloids and albumin. This led to a decrease in hematocrit (35.5 +/- 6.3-->31.8 +/- 5.6%, P < 0.01), fibrinogen, and platelets, as well as a decrease in fibrinolysis (Euglobulin Clot Lysis Time increases 246 +/- 52-->300 +/- 46 min and fast-acting plasminogen activator inhibitor 1 [PAI-1] activity increases 10.5 +/- 6.9-->15.1 +/- 9 IU/mL, P < 0.01). There was also specific rheologic impairment with a dissociation of erythro-aggregates (primary aggregation time 3.37 +/- 2.63-->7.18 +/- 7.2 s). Tissue-type plasminogen activator (t-PA) antigen was only increased just after surgery (8.3-->14.5 ng/mL, P < 0.01). During the first postoperative week, the acute-phase response subsided. This was accompanied by an increase in fibrinogen, von Willebrand factor antigen, and plasma viscosity (1.33 +/- 0.13-->1.49 +/- 0.13 mPa x s, P < 0.01). Hematocrit and the extrinsic fibrinolytic system (t-PA/PAI) returned to baseline values, whereas intrinsic fibrinolysis remained altered (the Euglobulin Clot Lysis Time, reflecting total activity of plasminogen activators, was still increased). Postoperative management may benefit from a recognition of these two distinct phases induced by surgery. The acute-phase reaction of the first postoperative week is an added vascular risk factor and requires a specific therapeutic approach.

Thrombomodulin (TM) is a very efficient natural anti-thrombin glycoprotein expressed on the endothelial cell surface. Circulating soluble thrombomodulin is also detected by enzyme immunoassay in plasma and represents some fragments of membrane TM with various molecular weight. Plasma TM (TMp) levels are elevated in diseases associated with endothelium damage. We have explored TMp in patients with atheromatous disease and compared its level with others endothelial cell markers, particularly those who indicate cell activation, as tissue-type plasminogen activator (t-PA), inhibitor of plasminogen activator (PAI-1) and prostacyclin (PG12). Thirty seven patients with documented atheromatous artery disease were included in this study. They were not diabetics and their hepatic and renal functions were normal. Mean age was 71 +/- years. Routine serum parameters were checked out as well as others more specific for endothelium activation (TMp, PG12, PAI-1, t-PA) measured by enzyme immunoassay. Patients were classified according to three localizations of atheromatous involvement: - 15 patients with peripheral occlusive arteriopathy disease (POAD) - 6 with coronary artery disease (CAD); and 16 with polyvascular involvement (POLY). They were compared with 21 controls without any vascular lesions (mean age: 43 +/- 13 years). In controls TMp was 36 +/- 8 ng/ml without significant change according with age and sex. In patients whatever the localization of atheroma, TMp was found significantly higher: POAD = 51.3 +/- 19.7 ng/ml (p = 0.003); CAD = 49.2 +/- 15.4 ng/ml (p = 0.008); POLY = 49.6 +/- 17.2 ng/ml (p = 0.003). A positive correlation was pointed out in all patients between TMp and t-PA (p = 0.047), TMp and PG12 (p = 0.008). A positive correlation between TMp and t-PA (p = 0.034) was found only in the subgroup with POAD. In this study, there was no correlation between TMp and the following parameters: leucocytes, haemoglobin, cholesterol, HDL, LDL-cholesterol, Lp(a), fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

A method for the detection of poly(A)+ RNA in cell suspensions by in situ hybridization and flow cytometry is described. The hybridizing properties of oligothymidylate o(dT) was well preserved after coupling to fluorescein. This fluorescent oligonucleotide was used as a probe to determine the poly(A)+ RNA content of fixed HL60 leukemic cells by flow cytometry. Labeling was considerably reduced by treating the cells with RNAse, and by competitive hybridization with either poly(U) or free poly(A). Labeling was also decreased in a time-dependent fashion by incubating the cells with actinomycin D prior to fixation. This method represents an improvement on the methods measuring total RNA and could be of value in investigations on the effect of drugs on RNA metabolism.

Some derivatives of thieno[2,3-d]pyrimidin-4(3 H)-one A and their 1,2-dihydrogenated homologues B were synthesized. The study of their in vitro antiaggregating activity showed that the first compounds exhibited significant inhibiting power when aggregation was induced with ADP. Their reduction to derivatives of 1,2-dihydrothieno[2,3-d]pyrimidin-4(3 H)-one led to a new series of molecules possessing a large antiaggregant activity. When compared to that of acetylsalicylic acid under the same conditions, this activity was the same with collagen or arachidonic acid-induced aggregation, but greater when aggregation was induced by ADP. Serotonin release was also inhibited.

Some new 4-quinazolinones were prepared. Their antiplatelet activity was evaluated in vitro with respect to aggregation induced by ADP, collagen, arachidonic acid and the platelet serotonin release reaction. Most molecules showed an inhibiting power similar to that of acetylsalicylic acid under the same conditions, and even greater when aggregation was induced by ADP. Reduction of the 4-quinazolinone derivatives to their 1,2,3,4-tetrahydroquinazoline homologues produced an increase in platelet inhibitory action except when ADP is the inductor.

Anthracyclines (ANT) are used in the treatment of leukemia and other cancers. These drugs have been shown to intercalate between the strands of DNA. In the present study, we show that the amount of ANT intercalated into DNA can be determined by measuring the fluorescence resonance energy transfer (FRET) between Hoechst 33342 (H33342) and ANT bound to DNA. The transfer efficiency was found to depend on the amount of disposable ANT but was independent of the amount of H33342 bound to DNA over a wide range of H33342 concentrations. The method was adapted for flow cytometric measurement of FRET in whole living cells and was used to evaluate the degree of intercalation of daunorubicin (DAU) and idarubicine (IDA) into DAU-sensitive and DAU-resistant leukemic cell lines. ANT intercalation into DNA was affected by factors which modify the intracytoplasmic concentration of ANT, and it was shown that the action of ANT and the resistance to ANT could not be attributed solely to the intercalative effect of the drugs. The method has advantages over previously described methods and represents a useful complementary tool in studies on the mode of action of ANT and the mechanisms of chemoresistance.

The relationship between heparin and fibrinolysis is strongly suggested. We have studied the influence on fibrinolysis of standard heparin (SH), Calciparin and low molecular weight heparin (LMWH), Fraxiparin, given preventively in an elderly population. Patients were randomized into two groups (SH, LMWH). We investigated fibrinolytic parameters (ECLT, t-PA antigen, PAI-1 activity and antigen, t-PA/PAI-1 complexes) before treatment (D0) and at D30 and D60, before and after venous occlusion (VO). Values at D0, D30 and D60 were compared within each group. A significant and marked increase in t-PA and t-PA/PAI-1 complexes at D30 and D60 before and after VO was noted only in the SH group. The mechanism and clinical relevance of this increase remains to be established.

Blood cell counts were performed on blood samples from 37 patients with imported malaria using three different blood analyzers (Coulter STKR, Coulter VCS and Technicon H1). Results were controlled by direct microscopic examination. Anemia, leukopenia, thrombocytopenia, or abnormalities of the leukocyte differential count were found in 32, 24, 30 and 92% of patients, respectively. The automatic analyzers gave alert messages for 70 to 75% of specimens, including specimens from ten patients with low parasitemias. These abnormalities should prompt careful analysis of blood smears when drug-resistance is suspected.

A variety of protein C assays are available as commercial kits. A collaborative study was undertaken to evaluate the performance of protein C assays. Various samples including calibrated plasmas and high purity concentrates destined to therapy were distributed among five laboratories. This comparison of protein C assays indicates that protein C levels measured by different functional or immunological assays and by five laboratories are very close for calibrated plasmas but not for high purity concentrates. The selection of the standard and the dilution buffer for protein C concentrates have important implications for the interpretation of the results. Dilution of purified protein C concentrates in protein C deficient plasma which restaure a total protein level similar to that of normal plasma improve the accuracy of functional protein C assays.

In vitro studies on the action of buflomedil (BFL) and its derivative CRL 41034 on the polymorphonuclear cells (PMN) has been performed using functional tests and scanning electron microscopy. The two drugs exhibited the same effects. BFL does not change the in vitro chemotaxis of PMN, but exhibits a regulatory effect on ZMS-induced aggregation of these cells. BFL also appeared to decrease superoxide production of PMN, in a dose- and time-dependent way. The cytoskeleton F-actin polymerization, analyzed through the binding of rhodamin-phalloidin, was increased when the total F-actin of the cells was unchanged. When cell extensions were studied morphologically a change in the shape of the pseudopods as well as the general aspect of the PMN (cottonous aspect) was observed as compared to controls. These drug-induced modifications in the shape change may be efficient in adhesion processes. Finally this latter effect and the influence on oxygen metabolite production could be another means of BFL to protect the microvessels during ischemia, in addition to its vasomotion promoting properties.

The activation of human polymorphonuclear cells (PMN) by the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), or the protein kinase C activator, phorbol myristate acetate (PMA), was studied using flow cytometry. Two probes were used to evaluate PMN activation: 1) a monoclonal antibody (MoF11) directed against an antigen (Ag) expressed on the membrane of monocytes and of activated PMN; 2) rhodamine phalloidin was used at the cytoplasmic level to measure the F-actin content. The expression of MoF11 antigen was found to be 3 to 5 times greater on the membrane of PMN activated by either FMLP or PMN as compared with membrane expression of the same Ag on resting PMN. This increase was found to be dose dependent for the two activators. Kinetic studies showed that a maximum response was observed in 1 to 2 min at 37 degrees C when FMLP was used, whilst a similar response required 10 min when PMA was used. The same discrepancy with activators was observed when actin polymerization was measured by labelling with rhodamine phalloidin. However, pretreatment of PMN with cytochalasin B inhibited actin polymerization whilst MoF11 antigen expression was increased, suggesting that the MoF11 antigen could be stored in granules of resting PMN. The study of actin polymerization and of MoF11 antigen expression, separately or in combination, could be a useful tool for the detection of activated PMN in biological samples.

The White Blood Cell differential given by the new analyser Coulter STKS was studied in a group of hospitalized patients originating from non-hematological departments (group 1; n = 290) and a group of patients suffering from an hematological disease (group 2; n = 106) with blood smear abnormalities. A good relationship between the reference method and the STKS results was shown in 200 patients without hematological abnormalities. Both sensitivity and reliability of alarms were evaluated in both groups. In group 1, we noticed respectively: 208 true negative, 40 true positive, 7 false negative, 35 false positive. Six of the seven false negative results were related to the presence of a slight myelemia without hematological significance, one to the presence of small quantity of circulating blasts with a leuconeutropenia. In group 2, all abnormalities seen on the blood smear were detected by the Coulter STKS either by an alarm message (93 cases) or by an increase of the lymphocyte population (9 cases) in some chronic lymphocyte leukemias, or by an important leuconeutropenia (4 cases) in acute treated leukemias. The results of the White Blood Cell differential performed with the Coulter STKS exhibited a good relationship with the reference method for normal bloods and a good reliability for detection of abnormal cells.

Pentoxifylline (PTX) has been reported to enhance the early accumulation of neutrophils at the site of Staphylococcus aureus subcutaneous infection in mice (1) and to stimulate in vitro PMN chemotaxis, particularly under dense agarose (2). Among the biochemical events contributing to chemotaxis are actin polymerization (3). The membrane cytoskeleton is believed to control the lateral mobility of integral membrane proteins as well as influencing cell shape and mobility. Thus, pharmacological modulations of neutrophil chemotaxis may be related to an effect of the pharmacological agents on the membrane cytoskeleton. The present study was designed to characterize the effect of PTX on actin polymerization of freely-suspended PMN before and after stimulation by the chemotactic factor f-MLP. We used flow cytometry to determine the proportion of actin in the filamentous form, and Rhodamine-Phalloidin as fluorescent probe (4). PTX decreased actin polymerization in response to stimulation by f-MLP. The reduction in F-actin by PTX was higher in the samples with higher activation ratios as compared with untreated PMN.

Protein synthesis was investigated in human platelets by measuring incorporation of radio actively labelled amino acids into trichloroacetic acid (TCA) precipitable material. Platelet polysomes were characterized by their sedimentation rate in a sucrose gradient. It was confirmed that platelets synthesize proteins in their cytoplasm and that a part of their polysomes are bound to the skeletal framework. A higher level of protein synthesis was found in a population of small platelets separated on Ficoll (2%-4%) density gradient. Moreover, small platelets contained more polysomes than larger platelets. These results show that small platelets are more active in protein turnover or de novo synthesis. These findings can be related to peripheral maturation of platelets.

The patient, a 76-year-old man, was referred with fever, large ecchymotic lesions and ulcerative laryngitis. Blood counts showed a hemoglobin of 11 g/100 ml, hematocrit of 31%, red blood cell count of 3.5 X 10(12)/1, white blood cell count of 6.8 X 10(9)/1 and platelet count of 16.0 X 10(9)/1. The differential count showed 17% neutrophils, 4% lymphocytes, 40% promyelocytes and 39% myeloblasts. The sternal marrow sample showed a marked hypercellularity. Of the cells, 80-85% were hypergranular promyelocytes, some of them showing bundles of Auer rods. No granulocytic maturation was observed. A few erythroblasts were present. A disseminated intravascular coagulation was observed (fibrinogen 0.85 g/l, factor V 18%, fibrin degradation products 640 mg/l). The serum creatinin was at 217 micromol/1 and the urea at 16.8 mmol/1. The treatment (daunorubicin, heparin, platelet transfusion) was unsuccessful and the patient died three days after entering hospital. The bone marrow karyotype by direct examination showed only normal metaphases (32 photographed). All the metaphases from the unstimulated blood 48-h culture (25 photographed) were clonal, showing the pattern 47,XY,del(11) (q23),t(15;17) (q24? q22?), +mar. The marker was '16 like' in size but its origin could not be determined (Figs. 1 and 2).

Hemorheologic disorders are a frequent finding in circulating blood during vascular diseases (arterial disease of lower limb, cerebrovascular accidents). They participate in thrombogenesis and tissue ischemia production, and also in microcirculatory disturbances as shown by behavior in microvessels of red cells with decreased hereditary deformability (sickle cell anemia). Active alterations in erythrocyte rheology have also been demonstrated during vascular diseases in relation to inflammation: cell-protein inflammatory reaction, action of leukocytes. Therapy should be adapted for these microcirculatory disorders by suitable specific clinical trials.

An unfractionated mucosal heparin preparation and a low molecular weight heparin fraction were randomly administered to rats subcutaneously. This injection was made on rats with experimentally induced venous thrombosis. At high doses (6.66 mg/kg) of both drugs, thrombus mean weight was reduced in comparison with a control group. At lower doses (2.22 mg/kg) these two drugs inhibited the growth of the thrombus. This inhibition was obtained with a lower dose (0.66 mg/kg) of the low molecular weight heparin fraction than of heparin. The low molecular weight heparin fraction (molecular weight 5,000 daltons) has a high anti-Xa activity (200 units) and a low activity determined by the USP assay (50 units).

A haemorheological analysis with measured blood filterability was carried out on 179 patients with cerebrovascular accidents. Reduced blood filterability appeared to be related to the clinical condition: time variation (two phase course), influence of complications and of fatal prognosis. The underlying mechanism of the haemorheological disorders involved three groups of factors: quantitative and qualitative abnormalities of plasma proteins, red cells disturbances and white cells activation, leading to both local and general hyperviscosity, producing a prothrombotic state and a defective microcirculation. The haemorheological treatments are of value to the patients although the relationship between hemodilution and the filterability remains to be exactly defined, as well as a better understanding of the action of the drugs.

Rabbits given a hypercholesterolemic diet (500 mg/day) for 6 months and then maintained for another 6 months on a normal diet were found to have developed fibrous lipidic lesions in the aorta. Although circulating platelet levels in these animals were normal there was a reduction in mean megakaryocyte ploidy. The high concentrations of megakaryoblasts in all the sedimentation fractions collected by the 'STAPUT' system suggested an increase in megakaryocyte turnover with activation of committed stem cells. In addition, other defects in maturation of megakaryocytes were observed, such as abnormalities in the demarcation membrane system and granule number. These data reveal that defects in megakaryocyte maturation and turnover may occur during the process of reparative fibrosis of the arterial tree following a period of moderate hypercholesterolemic diet in the rabbit.

The progression of primary thrombocythemia (PT) into acute leukemia was diagnosed in a 64-year-old man who was previously treated by hydroxyurea without alkylating agents or radioactive phosphorus. Such an event has only been reported in very rare cases. The blast cells had a lymphocytic morphology but were identified as promegakaryoblasts by the ultrastructural demonstration of platelet peroxidase. These data suggest that a megakaryoblastic transformation could occur in PT as had previously been reported in chronic granulocytic leukemia.

A dozen acetoxy benzene derivatives (mono- or diacetylated diphenols) were tested for inhibitory activity on platelet aggregation in human platelet rich plasma induced with collagen, ADP and arachidonic acid. All the compounds tested showed an activity, and the results emphasize the antiaggregant properties of diacetylated derivatives which are more potent than aspirin. Unlike aspirin, their action is not prevented by salicylate. However, the large inhibition of serotonin release and thromboxane synthesis localizes their activity in arachidonic acid metabolism.

Blood and bone marrow samples from 106 patients with Acute Myeloid Leukemia were reviewed independently by three observers according to the FAB proposals. Without a cytochemical stain, complete agreement occurred in 75% of cases. The major discrepancies were between M1 and M5 poorly differentiated (4 cases) and M2 and M4 (12 cases). These discrepancies disappeared with cytochemistry. Others non-concurrences were between M1 and M2 (4 cases) M2 and M6 (3 cases), M4 and M5 differentiated (2 cases), M2 and RAEB (1 case). To solve these latter discrepancies some points of the FAB classification require clarification.

This report describes two elderly patients with acute leukemia in which blast cells were undifferentiated with conventional light microscopy (L.M.) and cytochemistry. Blast cells were identified as belonging to the erythroblastic line by their ultrastructural features: glycogen deposits, lipidic vacuoles, cytoplasmic ferritin molecules and rhopheocytotic invagination. Moreover, blast cells were surrounding a central macrophage. Thus, these two patients had acute erythroblastic leukemia which differs from erythroleukemia (M6 of FAB classification) in which blast cells present myeloblastic characteristics.

800 mg of pentoxifylline 400 was administered daily to two groups of elderly patients (average age 80 years) with the aim of increasing cerebral efficiency. Fifty patients (Group I) were treated for 30 days and of these, 25 patients (Group II) were treated for a further, 60 days. The programme included a battery of psychometric tests (Rey's R.P.M. Recit of Barbizet, coupled image tests, immediate memory test) two geriatric scales (Geriatric Rating Scale and Nosie 30) and study of the deformability of red cells. Results at the 30th and 90th day were compared with pre-experimental results: corrected filterability was increased (Group I and II, P less than 0.05), memory tests showed significant improvement (Group I and II, P less than 0.005), geriatric rating scale showed no modification. There was no correlation between changes in corrected filterability and improvement in psychometric tests.

Merocyanine 540 (MC540) has been reported to bind hemopoietic cells specifically. In this study, MC540 was used as a probe for the cytofluorometric discrimination of hemopoietic cells. In PHA-stimulated lymphocytes or HL-60 cells induced to differentiate with DMSO, MC540 binding was increased in actively proliferating cells and undifferentiated cells as compared with the more differentiated cells of the same lineage. Mononuclear bone marrow cells exhibited a discrete distribution of MC fluorescence. Sorting a population with high MC540 fluorescence (MC+ population) produced a 9-14-fold enrichment of granulocyte-macrophage progenitors (CFU-GM), and a recovery of all S-G2M phase cells (BrdUrd/DNA analysis). Cytological examination of the sorted MC+ population confirmed the enrichment in immature cells from all lineages. Double-labeling experiments using MC540 and Hoechst 33342 on total bone marrow or peripheral blood cells confirmed that the MC+ population included all the cycling cells. The proportion of S-G2M phase cells in this MC+ population was 29.3 +/- 7.8 for 15 bone marrow samples and 16.3 +/- 6.8 for 10 blood samples. MC540 could therefore be used as a marker for human hemopoietic cells, and it represents a useful tool for investigation of hemopoiesis in normal or leukemic bone marrow.

A 36 year old woman developed two thromboses on aortic valve prosthesis. The first thrombus at the 14th week of pregnancy was treated with urokinase (2,000 U/kg/h) plus heparin (700-1,000 U/h) over 24 hours and normal wing kinetics were obtained. The second thrombus developed at the 36 th week of pregnancy when the patient was receiving calciparin, and only transient improvement was obtained with similar doses of urokinase hourly over 72 hours. Progressive worsening resulted in higher doses (4,000 U/kg/h) being given without heparin and the thrombus then resolved. The use of urokinase for the first time in this indication allowed therefore, on two occasions and without hemorrhagic complications the cure of this recurrent thrombosis on aortic prosthesis, and the birth, by caesarean, of a healthy baby.

Pronounced and direct relations exist between hemorheologic blood disease and microcirculation in disorders of red blood cells. Within this framework, sickle cell anemia appears as the typical hemorheologic disease clearly illustrating the hemorheology-microcirculation-thrombosis relation. In acquired diseases, particularly those vascular disorders most concerned, the relation is indirect: the hemorheologic disorder predisposes to plasma and cell occlusion of the vascular lumen, probably labile and pre-thrombotic but influencing microcirculation blood flow. The role of endothelial cells and vascular wall in the appearance and localization of this phenomenon is unknown at present. Pharmacologic interest is considerable in both cases, with emphasis on the use of drugs of both cellular but also intercellular and parietal activity.

Bone marrow karyotypes of 78 adult patients with acute myelogenous leukemia (AML) were studied at the time of diagnosis, with special reference to the Sakurai and Sandberg classification (NN, karyotype completely normal; AN, mixture of normal and abnormal metaphases in the karyotype; AA, totally abnormal karyotype). The results showed no difference in complete remission rate (CR) or in survival time between the NN and the AN groups, but highly significant differences between the AA group and the NN and AN groups (whether taken together or separately). When studying the relationship between age and survival time, we found that 59 years of age was a frontier between two homogeneous groups having quite different prognoses. The NN/AN/AA classification had a good prognostic value in patients under 59 years of age. In older patients, no correlation was found between the classification and complete remission rate or survival time.

Efficacy of a very low molecular weight heparin, CY 222, in the treatment of deep venous thrombosis of lower limbs was evaluated in a prospective clinical trial instituted in November 1984. CY 222 was administered as subcutaneous injections of 0.03 ml.kg-1 daily (750 anti-Xa U.kg-1.d-1) as 3 divided doses over a minimum of 10 days. Efficacy was rated as a function of clinical and phlebographic criteria. The group of 95 patients treated was a heterogenious one: 38% medical, 62% surgical, and 48% of the total group had partial interruption of vena cava previous to study. The period between first clinical manifestations of the deep thrombosis and therapy varied between one day and 3 months (mean: 1 1/2 days). Clinical symptomatology significantly and globally regressed in 88% of the patients. Comparisons between phlebographic findings at start and end of treatment are expressed using Arnesen's score (cf. table).

We carried out a series of laboratory investigation in 28 patients with prosthetic valve thrombosis (17 mitral and 11 aortic). The diagnosis was confirmed by TM and bidimensional echocardiograms and by cineradiography. The treatment consisted of UK (4,500 IU/kg/hour for 12 hours) and/or SK (initial loading dose of 500,000 IU, then 150,000 IU/hour for 10 hours). We measured fibrinogen, degradation products of fibrinogen-fibrin, plasminogen and antiplasmin. Two dimensional electrophoresis against anti-fibrinogen, anti-fragment-D and anti-fragment-E were employed for some samples. After thrombolytic therapy (SK: 18; UK: 7; SK-UK: 2; UK-SK: 1) complete clot removal was obtained in 21 cases (successful: group S), partial removal in 4 cases (improvement: group A) and failure in 3 cases (failure: group E). 3 patients died. Biochemical effects can be seen in group S and in group A. There were no changes in group E.