Evaluation of the platelet counting by Abbott CELL-DYN SAPPHIRE haematology analyser compared with flow cytometry.

The Abbot Cell-Dyn Sapphire is a new generation haematology analyser. The system uses optical/fluorescence flow cytometry in combination with electronic impedance to produce a full blood count. Optical and impedance are the default methods for platelet counting while automated CD61-immunoplatelet analysis can be run as selectable test. The aim of this study was to determine the platelet count performance of the three counting methods available on the instrument and to compare the results with those provided by Becton Dickinson FACSCalibur flow cytometer used as reference method. A lipid interference experiment was also performed. Linearity, carryover and precision were good, and satisfactory agreement with reference method was found for the impedance, optical and CD61-immunoplatelet analysis, although this latter provided the closest results in comparison with flow cytometry. In the lipid interference experiment, a moderate inaccuracy of optical and immunoplatelet counts was observed starting from a very high lipid value.

RAS and MTHFR gene polymorphisms in a healthy exercise-trained population: association with the MTHFR (TT) genotype and a lower hemoglobin level.

The aim of this study was to determine the frequencies of ACE (I/D), AGT (M235T), AT1R (A1166C) and MTHFR (C677T) polymorphisms in a well-defined (in regards to health and nutritional status and lifestyle) population of young, healthy, exercise-trained subjects (no. 100) from the Campania region of Southern Italy. We also investigated whether there was any correlation between these polymorphisms and biochemical, hematological and hemostatic parameters in this "low-risk" population. Gene polymorphisms were analyzed with the polymerase chain reaction and restriction enzyme analysis. Allele frequencies of the genotypes examined were in Hardy-Weinberg equilibrium and agree with those reported in the Italian population. No associations were found between ACE, AGT, AT1R gene polymorphisms and anthropometric, clinical and laboratory parameters. However, the MTHFR (C677T) polymorphism was significantly associated with lower hemoglobin plasma levels in TT vs. CC + CT females (p < 0.016). This report is the first to describe the frequencies of RAS and MTHFR gene polymorphisms in young, exercise-trained volunteers from Campania and to identify an association between the MTHFR gene polymorphisms and lower hemoglobin plasma levels in young healthy females.

Evaluation of the monocyte counting by two automated haematology analysers compared with flow cytometry.

The aim is to determine the monocyte count performance of the Bayer Diagnostics ADVIA120 and Coulter LH 750 automated haematology analysers and the results obtained by these two instruments were compared with those provided by Becton Dickinson FACScan flow cytometer using the combination of CD45/CD14 MoAb. Linearity and imprecision were also evaluated. The linearity of both instruments was good. Coulter LH 750 showed better precision (4.3%) than ADVIA 120 (9.0%) both within and between batch. A significant correlation (r = 0.973) was found between the LH 750 and the flow cytometry method, while a modest one was observed between the latter and the ADVIA 120 (r = 0.880). When comparing the percentage of monocytes by means of one-way anova and Tukey test, it was found that the LH 750 provided the closest results in comparison with flow cytometry, with no statistical difference between the means (mean difference MO% = 0.6); however the difference was statistically different between the ADVIA 120 and flow cytometry (mean difference MO% = -4.06). These data were confirmed by Altman-Bland and Deming regression analyses.

Intravenous prostaglandin E1 reduces soluble vascular cell adhesion molecule-1 in peripheral arterial obstructive disease.

OBJECTIVES: Elevated levels of soluble (s) vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1, pointing to activation of cells involved in vascular inflammation, have been previously reported in peripheral arterial obstructive disease (PAOD). We tested the hypothesis that intravenous prostaglandin E(1) (PGE(1)) treatment, which produces clinical benefits in this condition, might decrease such levels. METHODS: Ten subjects (age range 58 +/- 10 years, 6 male, 4 female) with characterized Fontaine stage IIa to IV PAOD (ankle/arm pressure index <0.96) were entered into a treatment protocol with twice daily intravenous infusions of PGE(1) (alprostadil) at 120 microg per day, repeated for 10 consecutive days. Preinfusion and postinfusion plasma samples were stored for blind enzyme immunoassays of soluble adhesion molecules and the fibrinolytic marker tissue plasminogen activator, type-1 plasminogen-activator inhibitor, and D -dimer. RESULTS: Estimates of severity of pain at rest, consumption of analgesics, magnitude of trophic lesions, remission to lower Fontaine stages, and favorable changes in the venoarteriolar reflex documented significant beneficial effects of the treatment. Significant (P <.01) pretreatment and posttreatment reductions of in all soluble markers explored were found. Particularly, sVCAM-1 exhibited a significant decrease after each infusion, which was sustained at the last day of treatment (from 854 +/- 214 ng/mL to 775 +/- 215 ng/mL across the first infusion, from 773 +/- 146 ng/mL to 680 +/- 110 ng/mL across the last infusion). CONCLUSION: Thus a global decrease of vascular cell activation appears to occur as a result of PGE(1) administration and may contribute to the observed clinical benefits in PAOD.

Exercise increases soluble adhesion molecules ICAM-1 and VCAM-1 in patients with intermittent claudication.

Adhesion molecules play a relevant role in the pathogenesis of vascular diseases. In 21 patients with intermittent claudication and 18 sex- and age-matched control subjects, we measured plasma levels of the circulating form of the adhesion molecules E-selectin, P-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) alongside von Willebrand factor (vWF), at rest, at maximally tolerated exercise and 5, 15 and 30 min after exercise. In controls, plasma sICAM-1 levels did not change with exercise, while in claudicants they increased from 285+/-15 to 317+/-16 ng/ml (p<0.01). Also for sVCAM-1 exercise did not modify plasma levels of sVCAM-1 in controls but increased it in claudicants from 671+/-45 to 751+/-47 ng/ml (p<0.05). Similarly, vWF did not change with exercise in controls, but increased in claudicants from 100+/-9% to 111+/-8% of value for pooled normal plasma (p<0.05). Exercise-induced changes in sICAM-1 negatively correlated with the maximal tolerated walking time, which is an index of disease severity. These findings indicate that, in claudicants, exercise is associated with increase in plasma levels of sICAM-1 and sVCAM-1.

Circulating levels of cytokines and their site of production in patients with mild to severe chronic heart failure.

BACKGROUND: Patients with chronic heart failure have elevated levels of proinflammatory cytokines; however, the mechanism for their increased expression and the site of their production are unknown. METHODS: Twenty-two patients with heart failure, New York Heart Association functional class II to IV, underwent hemodynamic evaluation and echocardiographic study. Blood samples for cytokine evaluation were performed in the ascending aorta, coronary sinus, inferior vena cava, and hepatic vein. Levels of tumor necrosis factor-alpha (TNF-alpha), its soluble receptors sTNF-RI and sTNF-RII, interleukin-6 (IL-6), IL-6 soluble receptor, soluble gp130, interleukin-2 soluble receptor, and soluble Fas were measured with enzyme-linked immunosorbent assay kits. RESULTS: IL-6 concentrations were higher in class IV patients than in class III patients, which in turn were higher than those in class II. TNF-alpha, sTNF-RI, and sTNF-RII were higher in class IV patients than in class III and II patients. Significant correlations were found between IL-6 concentrations and left ventricular end-systolic volume (r = 0.64; P <.001), pulmonary wedge pressure (r = 0.56; P <.01), and left ventricular ejection fraction (r = -0.56; P <.01). No correlation was found between TNF-alpha and its soluble receptors and left ventricular volumes or hemodynamic measures. Finally, no difference in cytokine concentrations was found among the different sample sites. CONCLUSIONS: Among inflammatory cytokines, IL-6 concentrations better reflect the hemodynamic derangement in patients with heart failure. No cardiac or gut production of cytokines occurs in patients with mild to severe heart failure.

Measurement of soluble adhesion molecules in primary Raynaud's phenomenon and in Raynaud's phenomenon secondary to connective tissue diseases.

Adhesion molecules play a role in the inflammation and pathogenesis of vascular diseases. In 13 patients with primary Raynaud's phenomenon, 19 with Raynaud's phenomenon associated with connective tissue disease, and 16 control subjects, we measured plasma levels of soluble forms of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, and von Willebrand factor. Patients with secondary Raynaud's phenomenon had plasma levels of soluble forms of intercellular adhesion molecule- 1, vascular cell adhesion molecule-1, E-selectin, and von Willebrand factor which were significantly higher than in those with primary Raynaud's phenomenon and controls, while no difference was observed between patients with primary Raynaud's phenomenon and controls. Within the group with secondary Raynaud's phenomenon, the strongest correlations were between soluble forms of intercellular adhesion molecule-1 and both E-selectin, (r=0.67, P<0.001) and von Willebrand factor (r=0.58, P<0.01). In none of the three groups were the levels of soluble adhesion molecules and von Willebrand factor changed by exposure of hands to cold, although all patients had a definite vasospasm. In conclusion, this study indicates that primary Raynaud's phenomenon is not associated with elevation of soluble adhesion molecules and von Willebrand factor. Prospective studies are now required to investigate the role of these molecules as predictors of secondary diseases.

In vitro effect of avarone and avarol, a quinone/hydroquinone couple of marine origin, on platelet aggregation.

We investigated the effect on human platelet aggregation of the naturally occurring quinone/hydroquinone couple, avarone and avarol. Avarone exerted antiplatelet activity both on platelet-rich plasma and, to a greater extent, on washed platelets. The quinone inhibited the platelet aggregatory process with all the agonists used. The highest inhibitory potency occurred with arachidonic acid or A23187 as stimulating agents. In the case of agonists such as adenosine 5' diphosphate, platelet-activating factor or U46619, the antiaggregatory effect was more pronounced on the second wave. Inhibition of the aggregatory process paralleled thromboxane B2 formation. Avarol also exerted antiplatelet activity, even though its inhibitory potency was much lower than that of avarone.

In vivo accumulation of 8-hydroxy-2'-deoxyguanosine in DNA correlates with release of reactive oxygen species in Fanconi's anaemia families.

The present study was aimed at verifying the occurrence, if any, of in vivo oxidative DNA damage in FA homozygotes, their parents and siblings. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) was measured, by HPLC/EC, in DNA from circulating blood leucocytes from FA homozygotes and their relatives and compared with a group of paediatric and adult healthy subjects. The population studied consisted of: (i) 15 FA homozygotes; (ii) 24 FA heterozygotes; (iii) 11 siblings. The 8-OHdG level in FA homozygotes was significantly higher with respect to age-matched controls, with a mean level of 33.3 +/- 6.8 (mean +/- SE) and 3.9 +/- 0.26 8-OHdG/10(5) dG respectively. The FA parents (heterozygotes) also displayed higher 8-OHdG levels relative to controls. The release of hydroxyl (.OH) and .OH-like radicals from leucocytes was determined by luminol-dependent chemiluminescence (LDCL) in a subgroup of FA homo- and heterozygotes, showing a very large in vivo formation of non-superoxide radicals. Chromosomal instability was also measured in the FA population. When relating either 8-OHdG or LDCL levels to spontaneous or diepoxybutane-induced chromosomal instability (S-CI and DEB-CI respectively), a significant correlation was observed between the 8-OHdG, LDCL and S-CI data. Within families a positive association was found between 8-OHdG levels in homozygotes and their related heterozygotes, suggesting segregation of the genetic defect(s) underlying the abnormal oxidative metabolism. The present study provides evidence for an in vivo pro-oxidant state in FA, in terms of excess formation of .OH and .OH-like radicals, and of DNA hydroxyl adducts. This finding appears to be shared by homozygotes and, to a lesser extent, by heterozygotes.

Determination of the kinetics of Na+/H+ exchange in platelets using the Coulter S-plus cell counter.

Cell swelling, as measured by electronic cell sizing, is a good indicator of the Na+/H+ exchange activation. In this study the kinetic properties of the Na+/H+ exchanger were determined with the aid of the Coulter S-Plus VI D haematological cell counter. Cell swelling was measured in platelets suspended in Na-propionate medium. The rapid entry and intracellular dissociation of propionic acid induces activation of the exchanger, and in turn the uptake of water by osmosis. The fractional volume increase measured by the Coulter S-Plus was dependent on the external Na-concentration, with Km = 86 mmol/l. Saturation was reached at a propionate concentration of 140 mmol/l. Inhibition by amiloride was dose-dependent with Ki = 24 mumol/l. The activity of the exchanger was not modified by ouabain. These data are generally consistent with those published in previous reports, and indicate that automated haematological analysers are appropriate for the study of this aspect of platelet function.

Haemostasis unbalance in Pugh-scored liver cirrhosis: characteristic changes of plasma levels of protein C versus protein S.

Liver cirrhosis leads to a protido-synthetic impairment that alters the levels of blood clotting factors and haemostasis. The aim of this study was to assess the alterations of haemostatic parameters in the evolution of liver cirrhosis scored according to Child's classification, with Pugh's modifications. Thirty-seven patients suffering from alcoholic and non-alcoholic liver cirrhosis, representing stages A5, A6, B7, B8 and C10, were tested for the main blood clotting parameters, i.e. prothrombin time, factor VII, partial activated thromboplastin time, fibrinogen, plasminogen, alpha 2-antiplasmin and physiological inhibitors [antithrombin III (ATIII), protein C (PC), protein S (PS)]. No variations were observed between substages A5 and A6 in any of the parameters, except for coagulation inhibitor levels. Most parameters showed a progressive decrease in stages B and C of the disease. The most significant alterations were found in the physiological coagulation inhibitors, with a sharper decrease in PC and AT III level and a lesser decrease in the level of PS through stages A5 and B8: this evidence could assume an important biological and diagnostic significance.

Evidence of a cold immunoglobulin M autoantibody against 78-kD platelet glycoprotein in a case of EDTA-dependent pseudothrombocytopenia.

Pseudothrombocytopenia is a phenomenon in which the electronic count shows spuriously low platelet counts in subjects with normal platelet levels. The mechanism of anticoagulant-dependent pseudothrombocytopenia appears to involve cold reactive agglutinins against platelet antigens. The authors report a case of EDTA-dependent pseudothrombocytopenia with evidence of a cold immunoglobulin M antibody against 78-kD platelet membrane glycoprotein (GP). Cell counts were performed by Coulter Counter S-Plus VI (Coulter, Hialeah, FL) in the following anticoagulants: EDTA, Na-citrate, and citrate-theophylline-adenosine-dipyridamole. Anti-platelet antibodies and platelet membrane GP antigens were assayed by an immunofluorescence technique as described by Van dem Borne in 1978. An immunoglobulin M/lambda anti-platelet antibody was found to react in serum as well as in plasma EDTA at room temperature, but not at 37 degrees C. This antibody appeared to be directed against GP78 membrane antigen because this antigen was not detectable by immunofluorescence in platelets collected in EDTA and Na-citrate anticoagulant, whereas a fluorescence signal was revealed in platelets collected in citrate-theophylline-adenosine-dipyridamole. This evidence was confirmed by platelet clumping inhibition tests in which target platelets were pretreated with anti-GP monoclonal antibodies. Clumping in the presence of pseudothrombocytopenia serum was inhibited by anti-GP78kD and anti-GPIIb/IIIa but not by anti-Ib. In this case, GP78 appears to be involved in platelet clumping, together with IIb/IIIa complex. The partial inhibition of the phenomenon observed in citrate-theophylline-adenosine-dipyridamole is probably related to a lower expression of the membrane antigens in platelets collected in this anticoagulant.

Characterization of hemoglobin lepore variants by advanced mass-spectrometric procedures.

We describe an analytical protocol for characterizing the molecular structure of hemoglobin (Hb) Lepore variants by using two different mass-spectrometric approaches. The first method consists of direct examination of the chromatographically separated hybrid globins by electro-spray mass spectrometry; the variant Lepore globin is identified through the accurate determination of its molecular mass. Alternatively, the anomalous globins are digested with trypsin and their structures are determined by fast atom bombardment mass-spectrometric analysis of the peptide mixture. The application of this procedure to the identification of Hb Lepore Boston and Hb Lepore Baltimore is described.

Morphological and functional abnormalities in platelets exposed in vitro to ioglicinic acid, a new ionic contrast medium.

The inhibitory effect on platelet function induced by several radiographic contrast media is still poorly understood. In this study platelet abnormalities caused by in vitro addition of ioglicinic acid, a new ionic contrast medium, were evaluated. The appearance of several granules similar to dense bodies associated with shape change and internal reorganization were detected by electron microscopy techniques. A functional study revealed a marked decrease in the aggregating response of platelets to adenosine diphosphate and calcium ionophore A23187, while aggregation in response to collagen was completely normal. It is suggested that ioglicinic acid induces platelet abnormalities related to the effect on calcium movements and that studies with this contrast medium may help the understanding of some basic events of platelet activation.

Protein balance during very-low-calorie diets for the treatment of severe obesity.

Very-low-calorie diets (less than 500 kcal/day; VLCD) are widely used for the treatment of severe obesity. We report the effects of such diets, consisting of proteins only or proteins and carbohydrates (CH), on nitrogen balance and protein nutritional status of morbidly obese patients. Cumulative nitrogen loss, serum albumin, transferrin, prealbumin (PA) and retinol-binding protein (RBP) concentrations, and plasma amino acid profile were determined in two groups of obese patients: 5 subjects (3 women, 2 men: BMI 55.3 +/- 2.2 kg/m2) subjected for 4 weeks to a protein VLCD (40 g protein + 2 g fat) and 7 others (4 women, 3 men: BMI 45.6 +/- 2.8 kg/m2) received for the same length of time a protein + CH VLCD (34 g protein + 26 g CH). Nitrogen balance was determined weekly whilst plasma and serum variables were measured on days 0, 3, 5, 10, 20 and 28 of treatment. Nitrogen balance did not significantly differ between the two groups of patients throughout the treatment. Serum PA and RBP concentrations decreased from day 5 and day 10, respectively, in both groups. Plasma amino acids showed a similar pattern in the protein and protein + CH groups. Alanine gradually decreased below baseline values; after a peak value on day 5, branched-chain amino acids (valine, leucine, isoleucine) returned to baseline values in both groups. In conclusion, in severely obese patients subjected to VLCD, nitrogen balance, labile protein concentrations and plasma amino acid profile are not significantly affected by adding CH to proteins.

[Hematologic characterization and analysis of genetic transmission in a case of double heterozygosity Hb Lepore/beta-thalassemia]. / Caratterizzazione ematologica e analisi della trasmissione genetica di un caso di doppia eterozigosi Hb Lepore/beta-talassemia.

Double heterozygosis condition for hemoglobin variants induce clinical syndromes known as intermediate thalassemias. Their diagnosis is often of certain difficulty because of their low frequency and heterogeneity of clinical expressions. We report a case of a 4 year child admitted to our medical center with a story of hepatosplenomegaly. An appropriate hematological study on patient's family permitted a diagnosis of double heterozygosis for Hb Lepore and beta-thalassemia. Results of hematological investigation are reported.