Expectations and implications of blood sparing with hemoglobin based oxygen carriers.

To gain insight into the degree to which hemoglobin based oxygen carriers may replace the need for transfusion in surgical patients experiencing blood loss, a simple mathematical model was developed. This model predicts the amount of blood sparing resulting from a bolus infusion of different doses of hemoglobin solution as a function of circulating hemoglobin half-life and degree of erythropoesis enhancement subsequent to treatment. The results of this analysis are consistent with published clinical data and imply that blood sparing increases with increasing oxygen carrier dose and half-life, as well as increasing levels of erythropoesis enhancement. The analysis also predicts that the total circulating hemoglobin content in patients infused with HBOC solutions may reach a minimum value up to ten days after treatment.

Evaluation and validation of virus removal by ultrafiltration during the production of diaspirin crosslinked haemoglobin (DCLHb).

Virus retention during ultrafiltration through A/G Technology filter cartridges was investigated to characterize the removal process and validate the degree of virus titre reduction during the filtration of red blood cell haemolysates performed as part of the production of diaspirin crosslinked haemoglobin (DCLHb). When viruses were suspended in phosphate buffered saline solution, retention was greater with larger sized viruses and smaller filter pore size. Virus titre was maintained at starting levels in the filter retentate circuit during the course of filtration, suggesting that the virus removal mechanism is predominantly size exclusion. Evaluation of specific processing variables indicated that the retention of phiX174 virus was increased in the presence of red blood cell haemolysate or at high membrane crossflow rates and transmembrane pressures, while the retention of EMC virus was less sensitive to variations in these parameters. Using these results to design a validation protocol, log reduction values of >7.9 were demonstrated for the retention of human immunodeficiency virus, pseudorabies virus and bovine viral diarrhoea viruses, 7.6 for hepatitis A virus, and 4.2 for porcine parvovirus. It was also shown that the retention of viruses was maintained during repetitive use of the same filter cartridge.

Validation of the heat treatment step used in the production of diaspirin crosslinked hemoglobin (DCLHb) for viral inactivation.

A series of experiments was performed to assess the ability of the heat treatment step used in the manufacture of diaspirin crosslinked hemoglobin (DCLHb) to inactivate viruses. In-process solutions (reaction mixtures after the crosslinking process) from six different manufacturing lots were used as test media in a 1:680 scaled down system in which the key process parameters used in the large scale production were duplicated. The inactivation of five different viruses (Bovine Viral Diarrhea Virus, Pseudorabies Virus, Human Immunodeficiency Virus 1, Porcine Parvovirus and Hepatitis A Virus) was evaluated. Each validation experiment consisted of spiking the solution at 37 degrees C with virus, heating to 74 +/- 1 degrees C over a period of 30 minutes, holding at 74 +/- 1 degrees C for 90 minutes and cooling from 74 +/- 1 degrees C to less than 10 degrees C over a period of 30 minutes. Duplicate experiments were performed with each of the viruses with the exception of Human Immunodeficiency Virus 1, for which three experiments were performed. In each experiment samples were removed before, during, and after heating for the purpose of determining virus titer and evaluating key process parameters. The results obtained from these experiments confirmed that the key process parameters in these experiments using the scaled down test system reproduced those of the large scale manufacturing process. The results of the virus assays showed at least a 7 log reduction was accomplished by the heat treatment for each of the viruses tested.

Validation of the heat treatment step used in the production of diaspirin crosslinked hemoglobin (DCLHb) for viral inactivation--effect of crosslinking.

Two experiments were performed to assess viral inactivation during the crosslinking and heat treatment steps of the DCLHb manufacturing process. Stroma free hemoglobin (SFHb) collected from a large scale manufacturing lot was tested in a 1:680 scaled down system in which the key parameters used in the manufacturing process were replicated. In the first study Porcine Parvovirus (PPV), a non-enveloped virus, was used to assess inactivation, while in the second study Bovine Viral Diarrhea Virus (BVDV), an enveloped virus, was utilized. In both experiments, the SFHb solution was deoxygenated and an aliquot of virus suspension was added. To initiate the crosslinking reaction, a solution of bis (3,5-dibromosalicyl) fumarate (DBBF) in HEPES buffer was added to the test solution. In both experiments the reaction times and the degree of crosslinking were normal. After crosslinking, the reaction mixtures were heated to 74 +/- 1 degrees C over 30 minutes, held at 74 +/- 1 degrees C for 90 minutes, and cooled to less than 10 degrees C over 30 minutes. In each experiment the degree of crosslinking of final product was 100% and yield of hemoglobin recovery was normal. Samples were removed prior to crosslinking, after crosslinking and before, during and after heat treatment for determination of virus titer and evaluation of key process parameters. The results from these experiments were consistent with those obtained from the full scale manufacturing process for the deoxygenation, crosslinking and the heat treatment step during the production of DCLHb. The results of virus assays showed that crosslinking has no effect on viruses and their subsequent inactivation by heat treatment.

Detection of HLA IgG antibodies by two enzyme-linked immunoassays, solubilized HLA class I and PRA-STAT. Comparison with the AHG PRA.

HLA class I-directed IgG antibodies have traditionally been detected with a complement-dependent lymphocytotoxicity (CDL) technique. We have evaluated two solid-phase enzyme-linked immunoassays (EIA) and compared them with the CDL antihuman globulin (AHG) dithiothreitol-treated (DTT) PRA in their ability to discriminate between the presence or absence of HLA class I-directed IgG antibodies in serum from patients awaiting transplantation. The EIA were: (1) an EIA that uses solubilized HLA class I antigens (sHLA-I) isolated from a 240-member platelet donor pool, and (2) the PRA-STAT ELISA kit. For the first comparison, we used 691 serum samples from 272 patients taken before they had been transplanted. The data show a significant (P < 0.0001) linear correlation (r = 0.77 between the AHG DTT PRA and the sHLA EIA. They also demonstrate that the mean sHLA-I EIA ratio significantly increases (P < 0.01) above background levels with each stepwise increase in AHG DTT PRA level. Discordant results were 1.0% (7/691) for sHLA-I EIA+ PRA- and 6.3% (44/691) for PRA+ sHLA-I EIA-. However, a lower correlation was noted between the AHG DTT PRA and the PRA-STAT (Nextran) PRA results (n = 230; r = 0.42). The sHLA-I EIA is able to determine whether or not HLA Class I IgG antibodies are present in serum from transplant candidates and is an appropriate adjunct to the traditional CDL PRA assay, whereas the PRA-STAT is not.

Resuscitation with Diaspirin Crosslinked Hemoglobin in a pig model of hemorrhagic shock.

The efficacy of Diaspirin Crosslinked Hemoglobin (DCLHb) as a resuscitative fluid in hemorrhagic shock was compared to another colloid solution (human serum albumin, HSA) and a crystalloid solution (Lactated Ringer's, LR). Hemorrhage (35 mL/kg) was followed by isovolemic exchange then volume replacement. This modeled the clinical situation where resuscitative fluids are administered prior to stopping the hemorrhage, the hemorrhage is stopped, then blood volume is restored. Four combinations of resuscitative fluids were evaluated during isovolemic exchange: volume replacement: DCLHb:LR, HSA:LR, HSA:HSA and LR:LR. All doses were 10 mL/kg:35 mL/kg except LR:LR which was 10 mL/kg:125 mL/kg. Volume replacement was followed by a stabilization period and reinfusion of shed blood (35 mL/kg). MAP increased most rapidly using DCLHb (from 48 to 102 mmHg after 10 min of isovolemic exchange) and was maintained for at least 2 hours. Arterial oxygen content and acid-base status were significantly improved after resuscitation with DCLHb:LR vs. other resuscitative therapies. In conclusion, DCLHb:LR was an effective resuscitative therapy in treatment of hemorrhagic shock.

Preparation and characterization of diaspirin cross-linked hemoglobin solutions for preclinical studies.

During 1990 and 1991 the capability for repetitive, consecutive production of DCLHb solution to meet a rigorous and complete set of product criteria was demonstrated. In addition, through periodic monitoring of product stored under controlled conditions, the stability of all lots of DCLHb solution during frozen storage was demonstrated for more than a year. In this way, assurance was provided that the DCLHb solution used in preclinical testing met all product criteria throughout the biological testing period.

Trace element analyses of diaspirin cross-linked hemoglobin solutions.

A sensitive assay using inductively coupled plasma atomic emission spectroscopy (ICP-AES) has been applied to the measurement of trace elements in diaspirin cross-linked hemoglobin (DCLHb) solutions. Calcium, magnesium, zinc and iron were the only elements detected at greater than background levels. Ag, Al, As, B, Ba, Bi, Cd, Co, Cr, Cu, Mn, Mo, Pb, Sb, Se, Si, Sr, Ti, and V were present at concentrations either equal to the acid blanks or were not detected. Detection of non-heme iron (not incorporated into the hemoglobin porphyrin ring) in a 10 g/dL hemoglobin solution required the development of a special protocol. In this protocol a chelator, DTPA, was added to hemoglobin solutions to complex with both free and non-specifically bound non-heme iron. The resulting iron:DTPA complexes were separated from the hemoglobin molecules by ultrafiltration and the ultra-filtrate analyzed by ICP-AES. A modification of this assay in which the DTPA was omitted was used to measure the free non-heme iron in solution. Typical concentrations of chelatable (free and non-specifically bound) and solution (free) non-heme iron in DCLHb production lots at the completion of manufacture were 0.5-1.0 ppm and 0.1-0.3 ppm, respectively.

Effects of intravenous infusions of diaspirin cross-linked hemoglobin (DCLHb) on sheep.

The purpose of this study was to compare the cardiopulmonary, hematologic, and immunologic responses of unanesthetized sheep to single, "topload", intravenous infusions of either 10 mL/Kg or 40 mL/Kg of Diaspirin Cross-Linked Hemoglobin, 10 mL/Kg or 40 mL/Kg of a Human Serum Albumin (HSA) solution oncotically adjusted with human serum albumin to approximately match the oncotic pressure of the DCLHb, or 10 mL/Kg of Erythrocyte Hemolysate solution prepared in a manner similar to that commonly described in the literature and referred to as "stroma free hemoglobin". Solutions were infused at a rate of 1 mL/Kg/minute and animals were monitored for 72 hours after infusion. These studies demonstrated that in sheep infusion of either DCLHb or HSA solutions was well tolerated and did not produce a significant increase in plasma C3a levels, an increase in the plasma concentration of thromboxane B2, or unexpected fluid shifts. In contrast, infusion of the Erythrocyte Hemolysate produced a greater than 10-fold increase in plasma C3a concentrations, a greater than 6000-fold increase in plasma TxB2 concentration, significant fluid shifts, and changes in a variety of other parameters consistent with induction of a dramatic inflammatory response. These results indicate that appropriately prepared and purified DCLHb solutions do not elicit an inflammatory reaction in sheep.

Immunogenicity of diaspirin cross-linked human hemoglobin solutions.

To assess the potential immunogenicity of human diaspirin cross-linked hemoglobin (DCLHb) solution, repetitive doses of this material were given intravenously to rhesus monkeys at monthly intervals and the immune response to this challenge was assessed. Serum samples collected at multiple intervals throughout the study showed no evidence of DCLHb specific IgG or IgM production. Intradermal skin tests performed one month after the final DCLHb infusion were also negative. These data demonstrate that DCLHb is not antigenic when administered intravenously to rhesus monkeys. In addition, screening of a panel of normal human sera for pre-existing anti-DCLHb IgG antibodies was negative, suggesting that this modified hemoglobin is unlikely to be antigenic in humans.

The effect of the plasticizer di-2-ethylhexyl phthalate on the survival of stored RBCs.

Recent in vitro studies have shown that di-2-ethylhexyl-phthalate (DEHP) inhibits the deterioration of RBCs during refrigerated storage in containers that use this compound as a plasticizer. The experiments described in this report were designed to assess whether this in vitro protective effect of DEHP would result in a prolonged in vivo survival of RBCs infused into normal human recipients. Whole blood collected from ten normal donors was stored for 35 days in citrate-phosphate-dextrose-adenine (CPDA-1) anticoagulant contained in polyvinylchloride (PVC) bags plasticized with DEHP or a trimellitate compound that is known to have low leachability. Aliquots of RBCs from each container were then labeled with chromium-51 and were reinfused into the original donors. For blood stored in DEHP-plasticized PVC bags, 24% more red cells survived in vivo 24 hours after reinfusion than was observed when the blood had been stored in trimellitate-plasticized bags (P less than .001). Whole blood stored in glass bottles showed a similar improvement in in vivo survival when DEHP was added in weekly increments to mimic the accumulation of this plasticizer seen during storage in plastic containers. Survival of packed red cells stored in the presence of DEHP increased by 14% compared with storage in trimellitate-plasticized bags (P less than .05). In agreement with previous studies, hemolysis and microvesicle formation were also reduced in the presence of DEHP. These results suggest that proposed new storage systems lacking DEHP should be carefully evaluated to determine whether adequate post-transfusion survival of RBCs may be achieved.

Virus removal or inactivation in hemoglobin solutions by ultrafiltration or detergent/solvent treatment.

Two procedures to eliminate virus infectivity from hemoglobin solutions at ambient temperature were evaluated. In the first, virus removal was assessed during the ultrafiltration of hemoglobin solutions through a membrane with a nominal molecular weight cut-off of 100,000 Daltons. The results of this study demonstrated that less than 0.1% of any virus originally spiked into the solution was detectable in the ultrafiltrate. In the second procedure the inactivation of viruses in hemoglobin solutions incubated with tri(n-butyl)phosphate mixed with sodium cholate was studied. Greater than 99% of each of the enveloped viruses tested was inactivated during the first 15 minutes of incubation with greater than 10(5) plaque forming units/ml of each being inactivated after one to six hours. No inactivation of the non-enveloped poliovirus was effected by this treatment. The data imply that both ultrafiltration and detergent/solvent incubation may reduce virus infectivity in hemoglobin solutions, but neither method yields a completely virus free product.

Virus inactivation in hemoglobin solutions by heat.

To determine the feasibility of heat treating hemoglobin solutions to inactivate viruses, we performed experiments on the thermal stability of this protein and found that the structure and function of deoxyhemoglobin are well preserved during incubation at 60 degrees C for 10 hours at a pH of 7.5. Model viruses and the human immunodeficiency virus were rapidly inactivated under these conditions. The results imply that incubation at 60 degrees C is a practical method for inactivating viruses in hemoglobin solutions.

Optimization of erythrocyte membrane glycoprotein fluorescent labeling with dansylhydrazine after polyacrylamide gel electrophoresis.

An improved procedure for the labeling of glycoproteins with dansylhydrazine subsequent to electrophoresis in polyacrylamide gels is reported. This procedure is derived from the work of Eckhardt et al. (1976, Anal. Biochem. 73, 192-197) and Weber and Hof (1975, Biochem. Biophys. Res. Commun. 65, 1298-1302) who showed that dansylhydrazine may be condensed with the aldehyde groups of oxidized glycoprotein carbohydrates and the resulting hydrazones reduced with dimethylamine borane and/or sodium borohydride. Using the known distribution of erythrocyte membrane glycoproteins as a benchmark the effect of variation of a number of process parameters was investigated and an optimal procedure identified. The procedure is shown to be relatively insensitive to moderate variations in reagent composition, pH, and time of incubation with dansylhydrazine solution or reducing agents. It is also shown that labeling patterns may be preserved in dried gels if dimethylsulfoxide is replaced or omitted from all of the process solutions and destaining is effected with 1 M sodium acetate, pH 5.6. While specifically developed for the labeling of erythrocyte membrane proteins, the procedure is demonstrated to be applicable to other glycoprotein containing preparations.

Characterization of erythrocyte quality during the refrigerated storage of whole blood containing di-(2-ethylhexyl) phthalate.

Di-(2-ethylhexyl) phthalate (DEHP) accumulates in blood brought into contact with materials utilizing this compound as a plasticizer. To determine whether this phthalate diester affects red blood cell integrity, we have compared cell morphology, plasma hemoglobin accumulation, micro-vesicle production, and the concentration of intracellular metabolites and electrolytes of erythrocytes from blood stored at 4 degrees C with and without DEHP. When sufficient emulsified DEHP was mixed with blood to give a final concentration of 300 micrograms/mL, plasma hemoglobin accumulation was reduced by an average of 70%, the percentage of cells exhibiting normal morphology was enhanced by at least 20-fold, and the volume of microvesicles released from red blood cells was reduced by 50% after 35 days of refrigerated storage compared to the values obtained from corresponding samples stored without added phthalate. Similar effects were observed regardless of whether blood was stored in nonplasticized polypropylene or tri-(2-ethylhexyl) trimellitate plasticized polyvinylchloride containers and with DEHP solubilized by a variety of emulsifiers. When 300 micrograms/mL DEHP was added to stored blood containing erythrocytes predominantly in the echinocyte conformation, many of the cells reverted to the normal discoid morphology. The addition of this quantity of DEHP to blood had no significant effect on the course of storage-induced changes in erythrocyte adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG), sodium or potassium concentrations. The data are consistent with the hypothesis that DEHP inhibits the deterioration of the red blood cell membrane that results from the refrigerated storage of whole blood.

Thermal behavior of stearoylsphingomyelin-cholesterol dispersions.

The thermotropic behavior of aqueous dispersions of stearoylsphingomyelin-cholesterol mixtures was examined by high-sensitivity differential scanning calorimetry. When less than 20 mol % cholesterol was mixed with the sphingomyelin and the samples were held at room temperature for 7-9 days before the initiation of calorimetric measurements, a sharp endotherm at 56-57 degrees C and a broad endotherm at 35-50 degrees C were observed. In addition, samples containing 15-20 mol % stearol exhibited a sharp endotherm at 43-45 degrees C. If samples were held at room temperature for less than 2 h before the initiation of calorimetric analysis, the 56-57 degrees C endotherm was usually not seen. Instead, a combination of broad and sharp endotherms over the range of 35-50 degrees C was observed. Occasionally, exotherms were also observed within this temperature range. These results, along with those from previous studies, imply that a cholesterol-rich phase coexists with a cholesterol-poor phase in which the sphingomyelin molecules may exist in two distinctly different gel states.

Thermotropic behavior of dipalmitoylphosphatidylcholine vesicles reconstituted with the glycoprotein of vesicular stomatitis virus.

The vesicular stomatitis virus glycoprotein reconstituted into dipalmitoylphosphatidylcholine (DPPC) vesicles exerts a profound effect upon the DPPC gel to liquid-crystalline phase transition. The glycoprotein was reconstituted into DPPC vesicles by octyl glucoside dialysis. The gel to liquid-crystalline phase transition of these vesicles was monitored by differential scanning calorimetry. Vesicles formed in the absence of glycoprotein (600--2100-A diameter) underwent the phase transition at 41.0 degrees C and had an associated enthalpy change of 8.0 +/- 1.6 kcal/mol. Increasing the mole ratio of glycoprotein to DPPC in the vesicles to 0.15 mol % reduced both the transition temperature and the transition enthalpy change. The enthalpy change as a function of the mole percent glycoprotein could be fit to a straight line by a least-squares procedure. Extrapolation of the results to the glycoprotein concentration where the enthalpy change was zero indicated one glycoprotein molecule bound 270 +/- 150 molecules of DPPC.