Pietrina Piro

Alterations of red blood cell shape and sialic acid membrane content in septic patients

ObjectiveTo investigate the relationship between red blood cell (RBC) shape and modifications of RBC membrane protein content in critically ill patients with or without sepsis compared with healthy control volunteers. DesignProspective, observational in vitro study. SettingUniversity-affiliated cell biology laboratory. SubjectsHuman erythrocytes from healthy volunteers and nonseptic and septic intensive care unit patients. InterventionsSialic acid membrane content was measured on isolated RBC membrane proteins by high-performance liquid chromatography. RBC shape, estimated by the spherical index (M2/M1) or by the moment and effect of osmolality on RBC shape, was studied by flow cytometry at 25°C. Glycophorin A content was measured with antiglycophorin antibodies in flow cytometry. Measurements and Main ResultsSialic acid content was lower in the septic than in the nonseptic patients (1.98 ± 0.79, 2.20 ± 0.39 &mgr;g/100 &mgr;g membrane protein, respectively; p = .01) and than in the volunteers (2.71 ± 1.00 &mgr;g/100 &mgr;g membrane protein; p < .001). No significant difference was found in glycophorin A content between septic and nonseptic patients. RBCs from septic patients had a more spherical shape in isotonic solution than those of healthy volunteers, as assessed by a computed spherical index (M2/M1 ratio: 1.68 ± 0.34 vs. 1.95 ± 0.32; p = .001). Only the RBCs of septic patients failed to change their shape in hypo-osmolar solution (M2/M1 ratio: 1.68 ± 0.34 in iso-osmolar, 1.56 ± 0.28 in hypo-osmolar solution; p = .17). There was a significant correlation between the RBC shape evaluated by the spherical index or by the moment of the cytometric histogram and the sialic acid membrane content in all critically ill patients (septic and nonseptic patients) (r2 = .16, p = .01 for the moment, and r2 = .17, p = .01 for the spherical index, respectively). ConclusionsRBCs of septic patients are characterized by a more spherical shape, a decreased capacity of sphericity in hypo-osmolar solution, and a reduction in the sialic acid content of the RBC membrane. These modifications in RBC shape and membrane may contribute to the RBC rheologic abnormalities frequently described in sepsis.

Assessment of erythrocyte shape by flow cytometry techniques

Background: Red blood cell (RBC) rheology is altered in different diseases, including acute conditions such as patients in intensive care units (ICU) with sepsis or with an inflammatory reaction due to postoperative states or intracerebral haemorrhage, or chronic conditions such as diabetes mellitus or terminal renal failure. Several techniques are available to assess alterations in RBC rheology, especially deformability, but they are too cumbersome to be used on a large number of cells. Objective: To develop a new, rapid flow cytometry technique for easy assessment of RBC shape in patients. Methods: In flow cytometry, healthy human RBC shape shows a bimodal distribution related to the biconcave form. On this histogram, the second Pearson coefficient of dissymmetry (PCD) representing the asymmetry of this histogram and the spherical index (M2:M1) were calculated, both representing the spherical shape. This technique was used in healthy volunteers (n = 17) and in diseases characterised by abnormalities in RBC rheology, including terminal renal failure requiring haemodialysis (n = 28), diabetes mellitus (n = 18), sepsis (n = 19) and acute inflammatory states (postoperative, intracerebral haemorrhage, chronic obstructive pulmonary disease, epilepsy or severe drug intoxication; n = 21). Multivariate analysis was performed to determine the factors influencing RBC shape. Results: Measurement of RBC shape was highly reproducible. A good correlation was observed between the PCD and the spherical index, except in the critically ill patients without sepsis. RBCs were more spherical in patients with terminal renal failure (PCD −0.56 (0.14), p<0.05), diabetes mellitus (PCD −0.59 (0.23), p<0.05), sepsis (PCD −0.58 (0.22), p<0.05) or an acute inflammatory state (PCD −0.65 (0.29), p<0.05) than in healthy volunteers (PCD −0.89 (0.12)). The spherical index was also increased in all populations compared with healthy volunteers (terminal renal failure 2.30 (0.20); diabetes mellitus 2.27 (0.38); sepsis 2.28 (0.37); acute inflammatory state 2.35 (0.42) vs healthy volunteers 2.72 (0.47); all p<0.05). Multivariate analysis demonstrated that the underlying pathology (sepsis, acute inflammatory state, diabetes mellitus, terminal renal failure) was the principal cause of these RBC shape abnormalities. Conclusion: RBCs are characterised by an increased spherical shape in many disease states. The measure of the second PCD in flow cytometry is a new, easy method to investigate RBC shape in various diseases. This technique could facilitate the investigation of abnormalities of RBC rheology.

Monocyte-platelet complexes on CD14/CD16 monocyte subsets: relationship with ApoA-I levels. A preliminary study

The adhesion of the monocytes to the endothelium and their extravasation into the intima are key steps in atherogenesis. Studies showed the essential role of L-selectin (CD62-L), expressed by the monocytes, and the platelets by forming complexes with monocytes. The delipided apolipoprotein (Apo) A or high-density lipoprotein (HDL) has antiinflammatory effects on monocytes and can bind platelets (monocyte-platelet complexes [MPCs]). The aim of this study was to identify a possible relationship between the MPCs, the monocyte subset, and ApoA-I/HDL serum levels in vivo. Platelet-monocyte complexes were estimated by flow cytometry in 16 volunteers. Monocyte-platelet interaction was characterized by the percentage of monocytes coexpressing the constitutive platelet marker, glycocalicin gpIb-alpha (CD42b; CD42b+monocytes in %, MPC%). Monocytes were divided into four subsets based on lipopolysaccharide receptor (CD14) and FcgammaIII receptor (CD16) expression (CD14++/CD16-, G1; CD14++/CD16+, G2; CD14+/CD16-, G3; and CD14+/CD16+, G4). HDL and ApoA-I levels were measured by routine laboratory techniques. MPC% in the different subsets were G1=8.1+/-3.4%, G2=21.2+/-14%, G3=18+/-12.6%, and G4=22.3+/-14.3% (analysis of variance: P<.001). MPC% in the entire monocyte population was negatively correlated to ApoA-I (R=-0.71, P=.001). The relationship between ApoA-I and MPC% was found mainly in the subsets G1 (R=-0.67, P=.001) and G2 (R=-0.61, P=.01). MPC% was not correlated with any other lipids or lipoprotein or high-sensitivity C-reactive protein. When whole blood was incubated with HDL/ApoA-I, no modification of platelet CD42b fluorescence was observed, indicating that there is no direct interaction between the HDL/ApoA-I and the CD42b fluorescence. Among the monocytes, the G2 subset appeared to have the highest extravasation potential. Indeed, we previously showed that those cells overexpressed CD62-L, and we observed in this work that they were coated with platelets more than the G1 cells. The G2 subset could be more directly involved in the development of atherosclerotic lesions.

Effect of dipyridamole upon thymidine incorporation and capping in human lymphocytes

Dipyridamole is a potent inhibitor of tritiated thymidine incorporation by PHA-stimulated human lymphocytes. This effect is unrelated to the length of culture, to the level of response in untreated cultures, or to the proliferative index. This suggests that dipyridamole principally effects the membrane transport of thymidine. Dipyridamole inhibits sheep-erythrocyte-capping by E-rosettes. This effect cannot be mimicked by theophylline or cyclic nucleotides and cannot be reverted by adenosine. Pharmacological studies with colchicine and cytochalasin B suggest interference with cytoskeletal functions, probably of microtubules. This could be another site of action of dipyridamole beyond phosphodiesterase inhibition and adenosine metabolism.

Specific Effect of Age on Lectin-Induced Mitogenesis of Lymphocytes from Diseased Patients

Using several models of multivariate analysis, it is found that age per se is a specific determinant on the in vitro lymphocyte response to phytohemagglutinin A in a random sample of 30 diseased patients. Depending upon the used model, sex and lymphocyte subsets appear as other significant variables. The multiple regression coefficients reach 0.85. A 2% fall per decade is expected for phytohemagglutinin A responsiveness expressed in 1n cpm.

Thiobarbiturate and fructosamine assays: significance and interest of the borohydride blank

The acute-phase reaction (APR) induces the production by the liver of short-lived glycoproteins. The carbohydrate moiety of these proteins is thought to interfere with the thiobarbiturate (TBA) and nitroblue tetrazolium colorimetric tests which are used for assaying non-enzymatic glycosylation (NEG) of serum proteins. The aim of the present study was to assess the effect of the APR on the specificity of the colorimetric tests in non-diabetic and diabetic subjects. A positive correlation was found between C-reactive protein (CRP), an APR glycoprotein, and non-specific TBA reactivity as determined after borohydride reduction (BH4-resistant TBA, BR-TBA), both in non-diabetics (r=0.61;P<0.01) and diabetics (r=0.68;P<0.01). The BH4-sensitive specific TBA (SP-TBA) was not influenced by glycoproteins, and its increase in diabetics was correlated with the nitroblue tetrazolium assay (r=0.89;P<0.01). An independant effect of diabetes and APR on non-specific TBA was also demonstrated, suggesting an effect of hyperglycaemia on both protein glycation and glycosylation. TBA with borohydride reduction is an attractive tool for the study of complex glycoproteins in diabetes.