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As the lymphotropism of hepatitis C virus (HCV) has already been ascertained, and in the light of the fact that the immune defense system is an organized network composed of functionally interrelated tissues, this study was carried out to verify the possible involvement of spleen in HCV-related chronic hepatitis. In this cross-sectional study we measured spleen longitudinal diameter by ultrasound, beta2-microglobulin serum levels and splenic artery resistivity index (SARI) by Doppler in 51 patients treated with standard combined (Peg-Interferon plus Ribavirin) antiviral therapy. Thirty-three patients (17 females) completed the regimen and were compared to 31 controls (16 females). The mean basal values of spleen longitudinal diameter were higher in patients with chronic hepatitis than in controls, i.e., 116 mm (9.4) versus 102.7 mm (9.3), P = 0.0001. In the same patients a significant trend towards increased spleen longitudinal diameter was found after antiviral therapy, independently of the stage of HCV-related chronic hepatitis. The median values of the beta2-microglobulin concentrations were not significantly higher in the patients with HCV-related chronic hepatitis compared to controls, i.e., 1.3 (0.5 – 2.6) versus 1 (0.6 – 1.4), P = 0.16, although during the course of therapy they were significantly increased. SARI values of HCV-related chronic hepatitis patients were different from those of controls, but were unvaried compared to values at the end of treatment. Neither spleen measurements nor serum beta2-microglobulin levels were able to predict therapeutic response to antiviral therapy. A stimulation/ expansion of lymphoid tissue was found in patients with HCV-related chronic hepatitis. Such evidence raises the question whether physicians should continue to prescribe antiviral therapy in non-responders and supports the use of a new scheme (SLD plus β2-MG) to diagnose this ongoing, apparently reversible but nevertheless dangerous immunologic process.

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.

Summary The aim is to determine the monocyte count performance of the Bayer Diagnostics ADVIA®120 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.

A new generation hematology analyzer, Abbott CELL-DYN 4000 (CD 4000), capable of providing 26 parameters, including fully automated reticulocyte, nucleated RBC, blast, band, and immature granulocyte, and variant lymphocyte counts, was evaluated by using the National Committee for Clinical Laboratory Standards H20-A protocol and compared with the Bayer-Technicon H-2 analyzer, which is used routinely in our laboratory. A lipid interference experiment and a sample aging study also were performed. Linearity, carryover, and precision were within the limits established by the manufacturer, and satisfactory agreement was found with the H-2 analyzer. The evaluation of leukocyte differential counts indicated an excellent correlation with the manual reference method for neutrophils and lymphocytes, a good correlation for monocytes and eosinophils, and a poor correlation for basophils in samples with low counts; for basophil counts of 2% or higher, we found an improvement of the correlation coefficient. In the lipid interference experiment, only hemoglobin determination was influenced significantly on the CD 4000, but by using a new Abbott hemoglobin reagent, the interference was eliminated. The CBC and differential counts were stable and reportable up to at least 24 hours. Intrasample viability information on leukocytes provided a quality check on each individual specimen.

In this report we investigate the behaviour of the serum levels of Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) in the course of Systemic Sclerosis (SS). This cytokine is produced mainly by T and NK cells, and its possible role in the pathogenesis of SS has not been previously described in the literature. Serum GM-CSF levels were assayed in 10 female patients, ageing from 35 to 70, affected by SS. These patients were not suffering from other disorders and were not being treated with steroids or immunosuppressive drug. A solid phase immunoenzymatic method was used to assess the serum levels of GM-CSF. Reference values were previously determined in a control group of 36 healthy women blood donors (19 premenopausal and 17 postmenopausal) (x̄=20.1 ±12.3 pg/ml). All the patients but one showed significantly increased serum levels of GM-CSF (x̄= 120.9 ±125.5 pg/ml). The highest levels were found in the two oldest patients, who also had the longest clinical history of SS, but a clear correlation with age, disease duration or clinical manifestations was not evident, even if the postmenopausal age group patients showed a higher mean value of GM-CSF (x̄= 148.0±144.1 pg/ml) than that found in the premenopausal age group (x̄= 57.7±1.4 pg/ml) (in contrast with the findings in the control group). The absence of other pathogenic conditions in our patients suggests that the increase in serum levels of GM-CSF might be linked to the fibroblast proliferation which is typical of SS. However, our results do not explain the role played by this factor in the fibroblastic proliferation process and an in vitro study is necessary to clarify this aspect.

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.

Neonatal systemic infection is a leading cause of morbidity andmortality both in industrialized and developing countries.The increasing survival rates of preterm neonates with progres-sively lower gestational age and birth weight have been associatedwith higher occurrence of sepsis. Stratified by weight, approximately10% of 1000–1500 g infants and 35% of less than 1000 g babies willreceive a diagnosisof sepsis during their neonatalperiod. Early- onsetsepsis is acquired during the first 72 h of life via generally a verticalroute. Late-onset sepsis is much more common and infection comesgenerally from invasive devices and direct contact with parents andhealthcarepersonnel.Sepsisisresponsibleforroughly50%ofneonataldeaths after 2 weeks of life. Coagulase-negative staphylococci are theprevalent infecting organisms followed by E. coli and multidrugresistantGram-negativeorganismsincludingKlebsiella,Pseudomonasand fungi.Clinical presentation may start from subtle and unspecific signs(temperature instability, feeding intolerance, prolonged jaundice)and lead rapidly to catastrophic events such as death or permanentneurological impairment. Timely diagnosis is a greatly neededresearch goal. Current laboratory tests (CRP, total whites count,absolute neutrophil count, immature to total neutrophil ratio),individually or in combination, do not reach sufficient specificityand sensitivity. The diagnostic gold standard relies on the microor-ganism isolation, though generally only one out of five sepsisevaluation will result in a positive blood culture.Novel diagnostic tools for neonatal sepsisIn recent years a different approach has been attempted indiagnosing sepsis.The VCS technology of the Coulter LH 750 hematology analyzer(Beckman Coulter, Fullerton, CA) can obtain directly data from morethan 8000 WBCs using direct current impedance to measure cellvolume (V) for accurate size of all cell types, radio frequency opacityto characterize conductivity (C) for internal composition of each cell,and a laser beam to measure light scatter (S) for cytoplasmicgranularity andnuclearstructure.These datathen areused to identifyeach cell as a neutrophil, lymphocyte, monocyte, eosinophil, orbasophil, generating an automated differential count.However, the use of the VCS technology to evaluate morphologicchanges within the same cell population, such as the previouslydescribed neutrophil changes during acute bacterial infection, hasnever been well studied.Band forms and other immature granulocytes (metamyelocytes,myelocytes), as well as reactive segmented neutrophils, tend to belarger and have lower nuclear complexity than their normal“resting” counterparts. Therefore, Chaves et al. proposed that themorphologic changes seen in the left-shifted and reactivesegmented neutrophils could be analyzed quantitatively by usingthe Coulter LH 750 with VCS technology. These investigatorsevaluated the clinical usefulness of these morphologic parameters(also referred to as positional parameters) as possible indicators ofan acute infectious process. In a retrospective series of 69 adultseptic patients and 35 controls, the mean channel for meanneutrophil volume (MNeV) was significantly increased. With an150 MNeV cut-off, a specificity of 91% and a sensitivity of 70% wasachieved. A different series from the same research groupevaluated the usefulness of neutrophil volume distribution width(NDW). This parameter, too, was shown to correlate with apositive blood culture and an NDW cut-off of 23 produced aspecificity of 100% and a sensitivity of 69%.Our group conducted the first investigation using the VCStechnology in diagnosing late-onset neonatal sepsis. In a prospectiveseries of 120 very low birth weight newborns, a MNeV cut-off of 148produced a specificity of and sensitivity of in detecting late-onsetneonatal sepsis on a single determination. MNeV performed betterthananyother individualitem onthe rule outsepsispanel. NDWonlygave a specificity of 88% and a sensitivity of 95%.A larger, multicenter study is currently underway to validate theuse of positional parameters in screening for neonatal sepsis. Also,positional parameters have a potential role in detecting early-onsetsepsis and in guiding the length of antibiotic therapy. Due to theseverity of the disease, clinicians eagerly await an answer from thispromising field of investigation.

AIM: To shed some light on the relationship between anti-apoptotic serum Bcl-2 concentrations and metabolic status, anthropometric parameters, inflammation indices, and non-alcoholic fatty liver disease severity were investigated in 43 young individuals with fatty liver (FL) and 41 with nonalcoholic steatohepatitis (NASH). METHODS: Circulating levels of Bcl-2 were detected in 84 patients with ultrasonographic findings of "bright liver" and/or hyper-transaminasemia of unknown origin and/or increase in γ-glutamyl-transpeptidase (γ-GT) strictly in the absence of other acute or chronic liver disease, whose age was not advanced, who gave consent to liver biopsy and were then divided on the basis of the histological results into two groups (43 with FL and 41 with NASH). Twenty lean subjects, apparently healthy and young, were chosen as controls. RESULTS: Serum Bcl-2 concentrations were significantly higher in the FL group than in the NASH group. Insulin resistance and γ-GT activity were significantly higher in NASH subjects. Apoptotic hepatocytes were significantly more numerous in NASH patients. NASH patients presented with larger spleens and augmented C-reactive protein (CRP) concentrations than healthy subjects. Steatosis grade at histology was similar in both NASH and FL populations. The number of apoptotic cells was significantly related to anti-apoptotic Bcl-2 protein values in FL patients. Bcl-2 serum levels positively correlated to body mass index (BMI) values (P ≤ 0.0001) but not to age of the population. Triglycerides/HDL ratio correlated well to waist circumference in males (P = 0.0008). γ-GT activity was associated with homeostatic metabolic assessment (HOMA) (P = 0.0003) and with serum ferritin (P = 0.02). Bcl-2 concentrations were not related to either spleen size or CRP values. NASH patients presented a weak negative correlation between lobular inflammation and Bcl-2 levels. A prediction by low values of serum Bcl-2 towards a greater presence of metabolically unhealthy overweight/obese patients (MUOs) was evidenced. HOMA, BMI and uric acid, in that sequence, best predicted serum Bcl-2 concentrations. CONCLUSION: MUOs could be detected by Bcl-2 levels. By favoring the life span of hepatocytes, and enhancing triglyceride formation, the anti-apoptotic process inhibits free fatty acids toxicity in FL.

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.

In many platelet assays, as in measurement of platelet-adherent IgG (PAIgG), platelets are fixed in paraformaldehyde (PFA). To better clarify the effect of PFA on platelet size and on PAIgG measurement we compared PAIgG levels in a series of 40 samples, with or without PFA fixing. We used an ELISA which was set up on unfixed platelets and gave excellent results in terms of linearity (r = 0.923), precision (mean CV = 5%) and correlation with a platelet suspension immunofluorescence test. We found PAIgG values in unfixed platelets were about 10-fold higher than in PFA-fixed (0.411 +/- 0.172 fg/platelet vs. 0.035 +/- 0.019 fg/platelet). This discrepancy could be a consequence of the smaller mean platelet volume (MPV) of washed platelets when fixed in PFA (8.0 +/- 0.8 fl as compared to 10.1 +/- 1.07). This effect of PFA could decrease the amount of binding sites for IgG exposed on the platelet membrane and hence explain the significantly lower PAIgG values observed in fixed platelets. The PAIgG measurements on unfixed platelets from 200 healthy subjects displayed a Gaussian distribution with a mean +/- SD of 0.32 +/- 0.13 fg/platelet, i.e., 1200 +/- 500 molecules/platelet.

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.