A novel, reverse-phase-shifting, thermoreversible foaming hydrogel containing antibiotics for the treatment of thermal burns in a swine model - A pilot study.

BACKGROUND: This study compared a novel topical hydrogel burn dressing (CI-PRJ012) to standard of care (silver sulfadiazine) and to untreated control in a swine thermal burn model, to assess for wound healing properties both in the presence and absence of concomitant bacterial inoculation. METHODS: Eight equal burn wounds were created on six Yorkshire swine. Half the wounds were randomized to post-burn bacterial inoculation. Wounds were subsequently randomized to three treatments groups: no intervention, CI-PRJ012, or silver sulfadiazine cream. At study end, a blinded pathologist evaluated wounds for necrosis and bacterial colonization. RESULTS: When comparing CI-PRJ012 and silver sulfadiazine cream to no treatment, both agents significantly reduced the amount of necrosis and bacteria at 7 days after wound creation (p < 0.01, independently for both). Further, CI-PRJ012 was found to be significantly better than silver sulfadiazine (p < 0.02) in reducing bacterial colonization. For wound necrosis, no significant difference was found between silver sulfadiazine cream and CI-PRJ012 (p = 0.33). CONCLUSIONS: CI-PRJ012 decreases necrosis and bacterial colonization compared to no treatment in a swine model. CI-PRJ012 appeared to perform comparably to silver sulfadiazine. CI-PRJ012, which is easily removed with the application of room-temperature water, may provide clinical advantages over silver sulfadiazine.

Thermoreversible Reverse-Phase-Shift Foam for Treatment of Noncompressible Torso Hemorrhage, a Safety Trial in a Porcine Model.

INTRODUCTION: Noncompressible torso hemorrhage is the leading cause of exsanguination on the battlefield. A self-expanding, intraperitoneal deployed, thermoreversible foam has been developed that can be easily administered by a medic in austere settings to temporarily tamponade noncompressible torso hemorrhage. The purpose of this study was to assess the long-term safety and physical characteristics of using Fast Onset Abdominal Management (FOAM; Critical Innovations LLC) in swine. MATERIALS AND METHODS: Yorkshire swine (40-60 kg) were sedated, intubated, and placed on ventilatory support. An external jugular catheter was placed for sampling of blood. Continuous heart rate, temperature, saturation of peripheral oxygen, end-tidal carbon dioxide, and peak airway pressures were monitored for a 4-hour period after intervention (i.e., FOAM agent injection or a sham introducer without agent delivery). The FOAM agent was injected to obtain an intra-abdominal pressure of 60 mmHg for at least 10 minutes. After 4 hours, the animals were removed from ventilatory support and returned to their housing for a period of 7-14 days. Group size analysis was not performed, as this was a descriptive safety study. Blood samples were obtained at baseline and at 1-hour post-intervention and then on days 1, 3, 7, and 14. Euthanasia, necropsy, and harvesting of samples for histologic analysis (from kidneys, terminal ilium, liver, pancreas, stomach, spleen, and lungs) were performed upon expiration. Histologic scoring for evidence of ischemia, necrosis, and abdominal compartment sequela was blinded and reported by semi-quantitative scale (range 0-4; 0 = no change, 1 = minimal, 2 = mild, 3 = moderate, and 4 = marked). Oregon Health & Science University's Institutional Animal Care and Use Committee, as well as the U.S. Army Animal Care and Use Review Office, approved this protocol before the initiation of experiments (respectively, protocol numbers IP00003591 and MT180006.e002). RESULTS: Five animals met a priori inclusion criteria, and all of these survived to their scheduled endpoints. Two animals received sham injections of the FOAM agent (one euthanized on day 7 and one on day 14), and three animals received FOAM agent injections (one euthanized on day 7 and two on day 14). A transitory increase in creatinine and lactate was detected during the first day in the FOAM injected swine but resolved by day 3. No FOAM agent was observed in the peritoneal cavity upon necropsy at day 7 or 14. Histologic data revealed no clinically relevant differences in any organ system between intervention and control animals upon sacrifice at day 7 or 14. CONCLUSIONS: This study describes the characteristics, survival, and histological analysis of using FOAM in a porcine model. In our study, FOAM reached the desired intra-abdominal pressure endpoint while not significantly altering basic hematologic parameters, except for transient elevations of creatinine and lactate on day 1. Furthermore, there was no clinical or histological relevant evidence of ischemia, necrosis, or intra-abdominal compartment syndrome. These results provide strong support for the safety of the FOAM device and will support the design of further regulatory studies in swine and humans.

Thermoreversible Reverse-Phase-Shift Foam for Treatment of Noncompressible Torso Hemorrhage.

BACKGROUND: Noncompressible torso hemorrhage (NCTH) is a leading cause of traumatic exsanguination, requiring emergent damage control surgery performed by a highly trained surgeon in a sterile operating environment. A self-expanding, intraabdominally deployed, thermoreversible foam is one proposed method to potentially task shift temporizing hemostasis to earlier providers and additional settings. The purpose of this study was to assess the feasibility of using Fast Onset Abdominal Management (FOAM) in a lethal swine model of NCTH. METHODS: This was a proof-of-concept study comparing FOAM intervention in large Yorkshire swine to historical control animals in the established Ross-Burns model of NCTH. After animal preparation, a Grade IV liver laceration was surgically induced, followed by a free bleed period of 10 min. FOAM was then deployed to a goal intraabdominal pressure of 60 mm Hg for 5 min, followed by a total 60-min observation period following injury. RESULTS: At the end of the experiment, the FOAM agent was found to be distributed throughout the peritoneal cavity in all animals, without signs of iatrogenic injury. The FOAM group demonstrated a significantly higher mean arterial pressure compared with historical controls and a trend toward improved survival: 82% (9/11) compared with 50% for controls (7/14; P = 0.082). CONCLUSIONS: This is the first study to describe the use of a thermoresponsive foam to manage NCTH and successfully demonstrated proof-of-concept feasibility of FOAM deployment. These results provide strong support for future, higher-powered studies to confirm improved survival with this novel intervention.

Antibodies against polyethylene glycol in healthy subjects and in patients treated with PEG-conjugated agents.

In contrast to the accepted general assumption that polyethylene glycol (PEG) is non-immunogenic and non-antigenic, animal studies clearly showed that uricase, ovalbumin and some other PEGylated agents can elicit antibody formation against PEG (anti-PEG). In humans, anti-PEG may limit therapeutic efficacy and/or reduce tolerance of PEG-asparaginase (PEG-ASNase) in patients with acute lymphoblastic leukemia and of pegloticase in patients with chronic gout, but did not impair hyposensitization of allergic patients with mPEG-modified ragweed extract or honeybee venom or the response to PEG-IFN in patients with hepatitis C. Of major importance is the recent finding of a 22 - 25% occurrence of anti-PEG in healthy blood donors, compared with a very low 0.2% occurrence two decades earlier. This increase may be due to an improvement of the limit of detection of antibodies during the years and to greater exposure to PEG and PEG-containing compounds in cosmetics, pharmaceuticals and processed food products. These results raise obvious concerns regarding the efficacy of PEG-conjugated drugs for a subset of patients. To address these concerns, the immunogenicity and antigenicity of approved PEGylated compounds should be carefully examined in humans. With all these data in hand, patients should be pre-screened and monitored for anti-PEG prior to and throughout a course of treatment with a PEGylated compound. Finally, protein conjugates with the poorly immunogenic hydroxy-PEG sequence or other hydrophilic polymers are in early phases of development and may represent an alternative to immunogenic PEGylated proteins.

The effect of three hemostatic agents on early bone healing in an animal model.

BACKGROUND: Resorbable bone hemostasis materials, oxidized regenerated cellulose (ORC) and microfibrillar collagen (MFC), remain at the site of application for up to 8 weeks and may impair osteogenesis. Our experimental study compared the effect of a water-soluble alkylene oxide copolymer (AOC) to ORC and MFC versus no hemostatic material on early bone healing. METHODS: Two circular 2.7 mm non-critical defects were made in each tibia of 12 rabbits. Sufficient AOC, ORC or MFC was applied to achieve hemostasis, and effectiveness recorded. An autologous blood clot was applied to control defects. Rabbits were sacrificed at 17 days, tibiae excised and fixed. Bone healing was quantitatively measured by micro-computed tomography (micro-CT) expressed as fractional bone volume, and qualitatively assessed by histological examination of decalcified sections. RESULTS: Hemostasis was immediate after application of MFC and AOC, after 1-2 minutes with ORC, and >5 minutes for control. At 17 days post-surgery, micro-CT analysis showed near-complete healing in control and AOC groups, partial healing in the ORC group and minimal healing in the MFC group. Fractional bone volume was 8 fold greater in the control and AOC groups than in the MFC group (0.42 ± 0.06, 0.40 ± 0.03 vs 0.05 ± 0.01, P < 0.001) and over 1.5-fold greater than in the ORC group (0.25 ± 0.03, P < 0.05). By histology, MFC remained at the application site with minimal healing at the defect margins and early fibrotic tissue within the defect. ORC-treated defects showed partial healing but with early fibrotic tissue in the marrow space. Conversely, control and AOC-treated defects demonstrated newly formed woven bone rich in cellular activity with no evidence of AOC remaining at the application site. CONCLUSIONS: Early healing appeared to be impaired by the presence of MFC and impeded by the presence of ORC. In contrast, AOC did not inhibit bone healing and suggest that AOC may be a better bone hemostatic material for procedures where bony fusion is critical and immediate hemostasis required.

Ultrasonic parametric imaging of erythrocyte aggregation using the structure factor size estimator.

Ultrasound characterization of erythrocyte aggregation (EA) is attractive because it is a non-invasive imaging modality that can be applied in vivo and in situ. An experimental validation of the Structure Factor Size Estimator (SFSE), a non-Rayleigh scattering model adapted for dense suspensions, was performed on 4 erythrocyte preparations with different aggregation tendencies. Erythrocyte preparations were circulated in Couette and tube flows while acoustically imaged over a bandwidth of 9-28 MHz. Two acoustically derived parameters, the packing factor (W) and ensemble averaged aggregate size (D), predictably increased with increasing EA, a finding corroborated by bulk viscosity measurements. In tube flow, a "black hole" reflecting the absence of aggregates was observed in the center stream of some parametric images. The SFSE clearly allowed quantifying the EA spatial distribution with larger aggregates closer to the tube walls as the aggregation tendency was increased. In Couette flow, W and D were uniformly distributed across the shear field. Assuming that the viscosity increase at low shear is mainly determined by EA, viscosity maps were computed in tube flow. Interestingly, erythrocyte suspensions with high aggregabilities resulted in homogeneous viscosity distributions, whereas a "normal" aggregability promoted the formation of concentric rings with varying viscosities.

The effects of a soluble polymer and bone wax on sternal healing in an animal model.

PURPOSE: This study compares the effects of a soluble polymer hemostatic material and bone wax on sternal bone healing. DESCRIPTION: Median sternotomies were performed on 20 New Zealand White rabbits, and sufficient polymer (Ostene; Ceremed Inc, Los Angeles CA) or bone wax (Bone Wax; Ethicon Inc, Somerville, NJ) was applied to achieve bone hemostasis. After 6 weeks, sternal healing was assessed using roentgenograms, histology, and mechanical strength testing. EVALUATION: Roentgenograms revealed normal bone healing in the polymer-treated group and nonunion in the bone wax group. Histology showed normal bone healing in the polymer group, with fibrotic scar tissue and the absence of new bone formation in the bone wax group. Mechanical strength testing showed that polymer-treated sternal segments were twice as strong as those treated with bone wax. They had a significantly higher flexural strength (2.53 +/- 0.43 vs. 1.29 +/- 0.37 megapascal [MPa]; p < 0.001) and Young's modulus (0.315 +/- 0.056 vs 0.146 +/- 0.031 MPa; p < 0.001). CONCLUSIONS: The application of the polymer hemostatic material to the sternum resulted in significantly stronger union compared with the use of bone wax.

Infection rates and healing using bone wax and a soluble polymer material.

The effects of using a newly available water-soluble polymer bone hemostatic material in a contaminated environment were assessed in a rabbit tibial defect model. Infection rates and healing of polymer-treated bone were compared with the infection and healing of bone wax-treated bone and untreated controls after a bacterial challenge. Defects created in 24 rabbit tibias were treated with the polymer or bone wax, or left without a hemostatic agent. The defects were inoculated with Staphylococcus aureus ATCC-29213 (2.5 x 10(4) colony-forming units). After 4 weeks, all defects treated with bone wax were infected and osteomyelitis had developed, and none had evidence of bone healing. In the polymer and control groups, two defects in each group (25%) had osteomyelitis develop. The remaining six defects in each group (75%) showed no osteomyelitis and exhibited normal bone healing. The polymer-treated defects had a considerably lower rate of osteomyelitis and positive bone cultures compared with the bone wax-treated group. There were no differences between the polymer-treated and control groups in the rates of osteomyelitis, positive cultures, or bone healing. The use of a soluble polymer as an alternative to bone wax may decrease the rates of postoperative bone infections.

Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients.

BACKGROUND: Rapid clearance of poly(ethylene glycol)-asparaginase (PEG-ASNase) has been reported for up to one-third of patients treated for acute lymphoblastic leukemia (ALL), potentially rendering their treatment ineffective. A 25% occurrence of an antibody against PEG (anti-PEG) was previously reported in healthy blood donors. The objective of the study was to determine whether anti-PEG was associated with rapid clearance PEG-ASNase. METHODS: The investigation reanalyzed stored sera from pediatric patients enrolled in the ALL Berlin-Frankfurt-Muenster 2000 studies. Twenty-eight samples were selected to include 15 subjects with undetectable ASNase activity after receiving PEG-ASNase. Sixteen subjects treated with unmodified ASNase were also included, 8 with low ASNase activity. Sera were tested for anti-PEG using 2 techniques: 1) serology, by agglutination of PEG-coated red blood cells; 2) flow cytometry, by analysis of 10 microm PEG beads stained for bound immunoglobulins. RESULTS. Of the 15 sera from PEG-ASNase-treated patients with undetectable ASNase activity, anti-PEG was detected in 9 by serology and in 12 by flow cytometry. Anti-PEG was detected in 1 PEG-ASNase-treated patient with lower ASNase activity (123 U/L). No relation was observed between anti-PEG and serum ASNase activity for patients treated with unmodified ASNase. CONCLUSIONS: The presence of anti-PEG was very closely associated with rapid clearance of PEG-ASNase. Further comprehensive studies are warranted to fully elucidate the effect of anti-PEG on PEG-conjugated agents. Screening and monitoring for anti-PEG may allow identification of patients for whom a modified dosing strategy or use of a non-PEGylated drug would be appropriate.

A method to optimize PEG-coating of red blood cells.

Alloimmunization to donor blood group antigens remains a significant problem in transfusion medicine. A proposed method to overcome donor-recipient blood group incompatibility is to mask the blood group antigens by the covalent attachment of poly(ethylene glycol) (PEG) to the red blood cell (RBC) membrane. Despite much work in the development of PEG-coating of RBCs, there is a paucity of data on the optimization of the PEG-coating technique; it is the aim of this study to determine the optimum conditions for PEG coating using a cyanuric chloride reactive derivative of methoxy-PEG as a model polymer. Activated PEG of molecular mass 5 kDa was covalently attached to human RBCs under various reaction conditions. Inhibition of binding of a blood-type specific antiserum (anti-D) was employed to evaluate the effect of the PEG-coating, quantified by hemocytometry and flow-cytometry. RBC morphology was examined by light and scanning electron microscopy. Statistical analysis of experimental design together with microscopy results showed that the optimum PEGylation conditions are pH = 8.7, temperature = 14 degrees C, and reaction time = 30 min. An optimum concentration of reactive PEG could not be determined. At high polymer concentrations (>25 mg/mL) a predominance of type III echinocytes was observed, and as a result, a concentration of 15 mg/mL is the highest recommended concentration for a linear PEG of molecular mass 5 kDa.

Ostene, a new water-soluble bone hemostasis agent.

Traditional formulations of bone wax are composed largely of beeswax and are well known to interfere with bone healing and cause inflammatory reactions. Ostene, a newly available bone hemostasis agent made of water-soluble alkylene oxide copolymers, was evaluated. The soft tissue response to Ostene was compared with bone wax and a polyethylene control after implantation into the paravertebral muscles of three rabbits. After 2 weeks, Ostene elicited no fibrous response, the polyethylene elicited a thin (less than 0.5 mm) fibrous response, and the bone wax was encased in a fibrous capsule 0.6 to 1.0 mm thick infiltrated with inflammatory cells. The effects of Ostene were compared with bone wax in a femur defect model in eight rabbits. Ostene showed no evidence of an adverse response in the cortical defect site, medullary cavity, or the surrounding tissue at 4 and 8 weeks. In contrast, bone wax at both time intervals elicited a foreign body response consisting of fibrous tissue infiltrated by macrophages, giant cells, and lymphocytes at the sites of the bone defects. Bone wax also displaced the bone marrow and interfered with bone ingrowth into the defects. Ostene provides the clinician a water-soluble bone hemostasis material that does not demonstrate the adverse tissue response or the interference with bone healing seen with the use of bone wax.

Rheologic behavior of sickle and normal red blood cell mixtures in sickle plasma: implications for transfusion therapy.

BACKGROUND: Guidelines for transfusion in sickle cell disease usually define an upper hematocrit (Hct) limit of 0.30 to 0.35 to avoid blood hyperviscosity. In vitro viscosity studies of normal (AA) and sickle (SS) red blood cell (RBC) mixtures in buffer appear to confirm that this Hct limit is optimal for oxygen delivery to vascular beds as judged by the ratio of Hct to viscosity, with this ratio often termed "oxygen or RBC transport effectiveness." In the absence of plasma, however, effects due to RBC-RBC interactions mediated by plasma proteins cannot be assessed. STUDY DESIGNS AND METHODS: To investigate the optimal Hct-to-viscosity ratio of RBCs in plasma, the rheologic effects of Hct (0.20-0.40), the proportion of SS RBCs (0-100%), and shear rate (1-1000/sec) for mixtures of oxygenated and deoxygenated SS and AA RBCs were evaluated in sickle plasma at 37 degrees C. RESULTS: RBC suspension viscosity was shear-dependent (i.e., viscosity decreased with increasing shear rate) and increased with Hct and proportion of SS RBCs. An "optimal" Hct level (defined as a maximal of the Hct-to-viscosity ratio) was seen only at shear rates above 50/sec. At lower shear rates (e.g., 5/sec), where plasma-mediated RBC-RBC interactions predominate, any increment in Hct was offset by a proportionally greater increase in viscosity, thus leading to a lower Hct-to-viscosity ratio. CONCLUSION: These results indicate the importance of plasma-mediated RBC interactions and suggest that the benefits of transfusion may vary depending on local flow rates (i.e., shear rates) and organ-specific hemodynamics.

Effects of red blood cell aggregation on myocardial hematocrit gradient using two approaches to increase aggregation.

The normal transmyocardial tissue hematocrit distribution (i.e., subepicardial greater than subendocardial) is known to be affected by red blood cell (RBC) aggregation. Prior studies employing the use of infused large macromolecules to increase erythrocyte aggregation are complicated by both increased plasma viscosity and dilution of plasma. Using a new technique to specifically alter the aggregation behavior by covalent attachment of Pluronic F-98 to the surface of the RBC, we have determined the effects of only enhanced aggregation (i.e., Pluronic F-98-coated RBCs) versus enhanced aggregation with increased plasma viscosity (i.e., an addition of 500 kDa dextran) on myocardial tissue hematocrit in rapidly frozen guinea pig hearts. Although both approaches equally increased aggregation, tissue hematocrit profiles differed markedly: 1) when Pluronic F-98-coated cells were used, the normal transmyocardial gradient was abolished, and 2) when dextran was added, the hematocrit remained at subepicardial levels for about one-half the thickness of the myocardium and then rapidly decreased to the control level in the subendocardial layer. Our results indicate that myocardial hematocrit profiles are sensitive to both RBC aggregation and to changes of plasma viscosity associated with increased RBC aggregation. Furthermore, they suggest the need for additional studies to explore the mechanisms affecting RBC distribution in three-dimensional vascular beds.

Effect of enhanced red blood cell aggregation on blood flow resistance in an isolated-perfused guinea pig heart preparation.

The role of red blood cell (RBC) aggregation as a determinant of in vivo blood flow is still unclear. This study was designed to investigate the influence of a well-controlled enhancement of RBC aggregation on blood flow resistance in an isolated-perfused heart preparation. Guinea pig hearts were perfused through a catheter inserted into the root of the aorta using a pressure servo-controlled pump system that maintained perfusion pressures of 30 to 100 mmHg. The hearts were beating at their intrinsic rates and pumping against the perfusion pressure. RBC aggregation was increased by Pluronic (F98) coating of RBC at a concentration 0.025 mg/ml, corresponding to about a 100% increment in RBC aggregation as measured by erythrocyte sedimentation rate. Isolated heart preparations were perfused with 0.40 l/l hematocrit unmodified guinea pig blood and with Pluronic-coated RBC suspensions in autologous plasma. At high perfusion pressures there were no significant differences between the flow resistance values for the two perfusates, with differences in flow resistance only becoming significant at lower perfusion pressures. These results can be interpreted to reflect the shear dependence of RBC aggregation: higher shear forces associated with higher perfusion pressures should have dispersed RBC aggregates resulting in blood flow resistances similar to control values. Experiments repeated in preparations in which the smooth muscle tone was inhibited by pre-treatment with papaverine indicated that significant effects of enhanced RBC aggregation could be detected at higher perfusion pressures, underlining the compensatory role of vasomotor control mechanisms.

Graded alterations of RBC aggregation influence in vivo blood flow resistance.

Although the effects of red blood cell (RBC) aggregation on low-shear rate blood viscosity are well known, the effects on in vivo flow resistance are still not fully resolved. The present study was designed to explore the in vivo effects of RBC aggregation on flow resistance using a novel technique to enhance aggregation: cells are covalently coated with a block copolymer (Pluronic F-98) and then suspended in unaltered plasma. RBC aggregation was increased in graded steps by varying the Pluronic concentration during cell coating and was verified by microscopy and erythrocyte sedimentation rate (ESR), which increased by 200% at the highest Pluronic level. RBC suspensions were perfused through an isolated in situ guinea pig hindlimb preparation while the arterial perfusion pressure was held constant at 100 mmHg via a pressure servo-controlled pump. No significant effects of enhanced RBC aggregation were observed when studies were conducted in preparations with intact vascular control mechanisms. However, after inhibition of smooth muscle tone (using 10(-4) M papaverin), a significant change in flow resistance was observed in a RBC suspension with a 97% increase of ESR. Additional enhancements of RBC aggregation (i.e., 136 and 162% increases of ESR) decreased flow resistance almost to control values. This was followed by another significant increase in flow resistance during perfusion with RBC suspensions with a 200% increase of ESR. This triphasic effect of graded increases of RBC aggregation is most likely explained by an interplay of several hemodynamic mechanisms that are triggered by enhanced RBC aggregation.

Modulation of endothelial nitric oxide synthase expression by red blood cell aggregation.

The effects of enhanced red blood cell (RBC) aggregation on nitric oxide (NO)-dependent vascular control mechanisms have been investigated in a rat exchange transfusion model. RBC aggregation for cells in native plasma was increased via a novel method using RBCs covalently coated with a 13-kDa poloxamer copolymer (Pluronic F-98); control experiments used RBCs coated with a nonaggregating 8.4-kDa poloxamer (Pluronic F-68). Rats exchange transfused with aggregating RBC suspensions demonstrated significantly enhanced RBC aggregation throughout the 5-day follow-up period, with mean arterial blood pressure increasing gradually over this period. Arterial segments ( approximately 300 microm in diameter) were isolated from gracilis muscle on the fifth day and mounted between two glass micropipettes in a special chamber equipped with pressure servo-control system. Dose-dependent dilation by ACh and flow-mediated dilation of arterial segments pressurized to 30 mmHg and preconstricted to 45-55% of the original diameter by phenylephrine were significantly blunted in rats with enhanced RBC aggregation. Both responses were totally abolished by nonspecific NO synthase (NOS) inhibitor (Nomega-nitro-l-arginine methyl ester) treatment of arterial segments, indicating that the responses were NO related. Additionally, expression of endothelial NOS protein was found to be decreased in muscle samples obtained from rats exchanged with aggregating cell suspensions. These results imply that enhanced RBC aggregation results in suppressed expression of NO synthesizing mechanisms, thereby leading to altered vasomotor tonus; the mechanisms involved most likely relate to decreased wall shear stresses due to decreased blood flow and/or increased axial accumulation of RBCs.

Surface characterization of poly(ethylene glycol) coated human red blood cells by particle electrophoresis.

Recent studies have shown that the covalent attachment of poly(ethylene glycol), abbreviated as PEG, to the surface of human red blood cells (RBC) leads to masking of membrane antigenic sites and inhibition of RBC aggregation. The effects of PEG coating on the regions near the RBC glycocalyx were thus explored using cell micro-electrophoresis. Both linear (3.35, 18.5, 35.0) and an 8-arm 35.9 kDa reactive PEG were used; in one series, thick cross-linked coats were obtained using a branched PEG amine as a cross-linker. The results indicate marked decreases of RBC mobility (up to 90%) which were affected by polymer molecular mass and geometry. Since PEG is neutral and its covalent attachment is predominantly to primary amine groups, such decreases of mobility most likely reflect structural changes near and within the RBC glycocalyx rather than decreased surface charge density. Experimental data were analyzed using a theoretical approach which allows calculation of the thickness and friction of the polymer layers: (1) for linear PEGs, thickness increased and friction decreased with polymer mass; (2) compared to linear PEGs of similar molecular mass, thickness was less and friction was greater for the branched PEG; (3) cross-linked PEG coatings were more than 50 nm thick and were insensitive to changes of ionic strength. These observations are consistent with the aggregation behavior of PEG-coated RBC and indicate the usefulness of micro-electrophoresis methods for studies of covalently-attached polymers: the resulting calculated thickness and friction factors should be of value in achieving desired cellular surface characteristics or levels of cell-cell interaction.