Deformability of passive and activated neutrophils in children with Gram-negative septicemia.

Animal experiments suggest that obstruction of small vessels by polymorphonuclear neutrophils (PMNs) may contribute to the disturbed microcirculation in septic shock. The purpose of this investigation was to study deformability and volume of passive and activated PMNs and immature neutrophils in 15 children with Gram-negative septicemia and in 20 healthy children. Membrane cytoplasm tongues of neutrophils were aspirated by means of a micropipette system into 2.5-microm (diameter) pipettes for 60 s. Tongue growth was used as an indicator of deformability and the cell volume was calculated from the cell circumference before aspiration. Septicemic children showed higher percentages of immature neutrophils (38+/-13%) and activated PMN (12+/-5%) than healthy children (3+/-2% and 3+/-2%). In septicemic children, cellular volume of passive neutrophils decreased progressively with increasing maturity from myeloblasts (493+/-105 fL) to mature PMNs (346+/-29 fL) and the final tongue length in the micropipette increased from 2.7+/-1.1 to 8.5+/-1.8 microm during maturation. The final tongue length of activated PMNs was decreased by 60% compared with that of passive PMNs. The increased number of rigid activated and immature neutrophils may contribute to impaired microcirculation in septicemic patients.

Formation and relaxation of erythrocyte membrane tethers in micropipettes.

The red blood cell (RBC) membrane forms tethers in response to shear forces acting on tiny membrane points. Tether formation depends on viscous and elastic membrane properties and has been used as indicator of membrane fragility. A micropipette technique was used to study time dependent tether formation and tether relaxation of individual RBC. Point attached RBC were aspirated at a negative pressure of -5 mm H2O into a micropipette with an internal diameter of 7.8 microm. If tether formation occurred and the tether reached a length of approximately 16 microm, the pipette was carefully pulled back. The RBC left the orifice of the micropipette and the tether relaxed and pulled the main body of the RBC back to the attachment point. The relaxation of the tethers was exponential. The time constant for tether relaxation was 0.144 s which is similar to the time constant for recovery of entire RBC from extensional elastic deformation. Repeated tether formation and relaxation of the same RBC led to an earlier begin of tether formation and changed the behavior of tether growth, although the relaxation time did not change. We conclude that repeated tether formation decreases the resistance of the RBC membrane to form tethers upon given shear forces. Weakening of the membrane due to repeated plastic deformation may play a role in the membrane loss of circulating RBC during aging.

Effects of dietary supplementation of saturated fatty acids and of n-6 or n-3 polyunsaturated fatty acids on plasma and red blood cell membrane phospholipids and deformability in weanling guinea pigs.

The fatty acid composition of plasma cholesteryl esters, plasma phospholipids, red blood cell (RBC) membrane phosphatidylcholine (corresponding to the outer membrane leaflet), and phosphatidylethanolamine (corresponding to the inner membrane leaflet) was investigated in weanling guinea pigs fed with diets of cacao (saturated fatty acids), sunflower oil [n-6 polyunsaturated fatty acids (PUFA)] or fish oil (n-3 PUFA) for 20 wk. RBC deformation was measured by means of a cell-transit analyzer (filtration) and a cone-plate rheoscope. The contents of saturated fatty acids in plasma phospholipids and RBC membrane leaflets were similar in all three groups. Diets with sunflower oil resulted in a high content of linoleic acid in plasma cholesteryl esters and in the outer leaflet of RBC membranes. Fatty acids of fish oil were mainly incorporated in plasma phospholipids and in the inner leaflet of RBC membranes. The arachidonic acid content was high in all groups in the plasma phospholipids and in the inner leaflet. The n-6 and n-3 PUFA were mainly incorporated in the inner leaflet. In all groups the polyunsaturated/saturated fatty acid ratio and the total PUFA content were similar in the inner RBC membrane. The RBC filtration times and the RBC deformation indices were not affected by the dietary treatment.

Effects of group-A streptolysin O on neonatal and adult red blood cell deformability and haemolysis.

Neonates are more susceptible than adults to many Gram-positive and Gram-negative bacterial infections. Whereas group B streptococcus causes life-threatening infections in neonates, group A beta-haemolytic streptococcus infections rarely occur in neonates. To test the hypothesis that group A streptococcus may have different effects on neonatal and adult red blood cells (RBCs), haemolysis and deformability (rheoscope) of RBCs from adults, full-term and pre-term neonates were studied during 60 min incubation with 1 haemolytic unit (HU) mL-1 group A streptolysin O (SLO). SLO incubation of adult RBCs resulted in almost linearly increasing time-dependent haemolysis reaching 82%, whereas haemolysis of neonatal RBCs was below 60% after 1 h. After 60 min SLO incubation, RBC deformation was significantly (P < 0.05) more reduced in adults than in full-term and preterm neonates. An inverse overall relationship (r = 0.68) between SLO-induced haemolysis and RBC deformation was found after 60 min of SLO incubation. We conclude that SLO causes less haemolysis and less impairment of RBC deformation in neonates than in adults. The decreased RBC deformation of unhaemolysed RBC indicates that, before lysis, mechanical RBC membrane properties are altered by SLO.

Group B Streptococcus impairs erythrocyte deformability in neonates more than in adults.

Group B beta-haemolytic Streptococcus (GBS) may cause severe septic shock and death in neonates, whereas this is rarely the case in adults. As impaired red blood cell (RBC) deformability might disturb microcirculation in septic shock, the in vitro effects of GBS (1.7 x 10(8) cfu/ml) on RBC deformation (rheoscope) and haemolysis were studied in blood from preterm infants, term neonates, and adults. Furthermore, RBC deformation was studied in term neonates with GBS sepsis. RBC deformation at a shear stress of 4 Pa decreased significantly within 5 minutes of GBS incubation in preterm infants (-13%) and term neonates (-9%). In adults RBC deformation did not change during the first 15 minutes, but decreased significantly after 30 (-10%) and 60 minutes (-13%). In the term infants there was little further decrease in RBC deformation between 5 and 60 minutes of GBS incubation; RBC deformation in preterm infants decreased by 19% after 60 minutes compared with the preincubation values. RBC deformation in septic neonates was significantly decreased at shear stresses of 1, 2, and 3 Pa (-19%, -18%, and -9%). Sixty minutes of incubation of RBC from adults and neonates with GBS and without GBS resulted in haemolysis below 4%. It is concluded that neither neonatal nor adult RBC are haemolysed by GBS. In vitro, neonatal RBC deformability is more impaired than that in adults. This may contribute to the high risk of neonates for compromised microcirculation and circulatory shock as a result of GBS sepsis.

Lipid A binding to neonatal and adult red blood cells.

Lipid A is responsible for the endotoxic activities of gram-negative bacteria. Binding of lipid A (50 micrograms/ml) to RBC was studied using a passive hemolysis test. RBC from adults, cord and venous RBC from full-term infants and RBC from preterm infants were studied. Lipid A sensitized RBC were hemolysed with anti-lipid A and guinea pig complement. Hemolysis was expressed as hemoglobin concentration (absorbance at 546 nm) in the supernatant after centrifugation. 50 micrograms/ml lipid A did not increase spontaneous hemolysis (< 3%) after 60 min of incubation in any of the four groups. The passive hemolysis test did not result in additional hemolysis (5%) of umbilical cord RBC. RBC of preterm infants showed a significant increase in hemolysis (24%) after 60 min of incubation. In RBC of full-term neonates, increased hemolysis (14 and 46%) was detected after 30 and 60 min of incubation. Adult RBC hemolysed stronger (26 and 64%) after 30 and 60 min than neonatal RBC. We conclude that lipid A binds less to neonatal RBC compared to adults.

Reactive hyperemia of skin microcirculation in septic neonates.

Reactive hyperemia after 1 min of arterial occlusion was studied in back, thigh and heel skin of 40 preterm and full-term neonates using laser Doppler flowmetry. Twelve infants had clinical signs of septicemia, but normal laboratory tests at the time of fluxmetry. However, CRP, leukocyte count and the ratio of immature to total neutrophils increased during the following days and septicemia was confirmed by positive blood cultures (septic group). Seven neonates with clinical signs of septicemia had developed neither positive blood cultures nor laboratory signs (non-septic group). Fifteen were healthy neonates. In the septic neonates, time to reach maximal hyperemia, maximum post-occlusive hyperemia and recovery time of skin perfusion were increased significantly in back and thigh skin and the heal skin temperature was decreased when compared to healthy neonates. Healthy and non-septic neonates showed no significant difference in any of the parameters. We conclude that altered reactive hyperemia in the skin may be an earlier sign of neonatal septicemia than laboratory tests.

Chemotherapy with cytosine arabinoside in a child with Burkitt's lymphoma on maintenance hemodialysis and hemofiltration.

A case of Burkitt's lymphoma (stage IV) in an 8-year-old boy with end-stage renal failure due to hemolytic uremic syndrome is reported. The boy was treated by maintenance hemodialysis (HD) and hemofiltration (HF). During chemotherapy treatment with continuous cytosine arabinoside (Ara-C) infusion (100 mg/m2/d) for 7 days, concentrations of Ara-C and its metabolite uracil arabinoside (Ara-U) were measured in blood, dialysate, and filtrate. Ara-C levels were always below 200 ng/ml and were only qualitatively detectable in blood, dialysate, and filtrate. Ara-U levels were higher than 200 ng/ml after 18 h treatment and were measured quantitatively. Ara-U clearance during 3 h HD was 92 ml/min and the calculated mass removal 14.7 mg/3 h. In contrast, the Ara-U clearance during 3 h HF was 14 ml/min and the mass removal was 6.7 mg/3 h. Ara-C and Ara-U are eliminated by HD and HF in anuric patients. A continuous infusion of 100 mg Ara-C m2/d during HD or HF treatment did not result in a serum concentration above 200 ng/ml.

Effect of lipid A on the deformability, membrane rigidity and geometry of human adult red blood cells.

Lipid A is responsible for the activities of endotoxin and may cause circulatory failure and haemolysis. This study evaluated the effects of different lipid A concentrations on red blood cell (RBC) deformation (rheoscope), the aspiration pressure required to aspirate RBC into 3.3 microns pipettes, the membrane shear elastic modulus (i.e. membrane rigidity) and cellular geometry (micropipette system) after 15 min of incubation. Lipid A concentrations of 10 and 100 micrograms ml-1 of RBCs decreased RBC deformability by 26% and 39%, respectively. The aspiration pressure for RBCs into a 3.3 microns micropipette increased by 235% at a lipid A concentration of 10 micrograms ml-1 and by 586% at a concentration of 100 micrograms ml-1. The elastic shear modulus almost doubled at a lipid A concentration of 10 micrograms ml-1 and tripled at 100 micrograms ml-1. At a lipid A concentration of 100 micrograms ml-1, 37% of RBCs showed spicules. These echinocytes were less deformable than discocytes. Mean corpuscular volume, RBC volume and surface area were not affected by lipid A. We conclude that lipid A causes marked reduction of RBC deformability due to increasing membrane rigidity.