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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.

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.

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.