Toxicity of human hemoglobin solution infused into rabbits.

Aqueous human hemoglobin is being considered as an oxygen-transporting resuscitation solution. However, there are some indications that such solutions may be toxic. We report here that at a clinically significant dose (20% to 30% of the estimated blood volume) of hemoglobin solution (10 gm/dl), toxicity similar to that reported in humans is produced in rabbits. This toxicity is characterized by cardiac rhythm disturbances (25%) and coagulation abnormalities, manifested as intravascular thrombi. Hypoxia, tissue necrosis (brain 29%, liver 25%), and death (22%) were seen. Hypoxia appears to be the cause of death, because the arterial PO2 of rabbits that died was significantly lower (43 +/- 8 mm Hg) than that of rabbits that lived (80 +/- 12 mm Hg). We also report the effect of the method of preparation or degree of purity on the observed toxicity. Stroma-free red blood cell hemolysate, stroma-free hemoglobin, zinc-precipitated hemoglobin, and chromatographically purified hemoglobin were infused. Purification of these solutions ranged from removal of stroma to removal of all detectable nonhemoglobin protein components. In addition, the nonhemoglobin proteins were concentrated and infused into rabbits to compare their effects. All of these hemoglobin solutions induced similar toxicity for rabbits infused with stroma-free hemoglobin. The nonhemoglobin proteins, as well as human serum albumin, exhibited no toxicity. These results demonstrate that, at the clinically relevant dose, hemoglobin or something inherently associated with hemoglobin is toxic.

Synergistic toxicity of endotoxin and hemoglobin.

Stroma-free hemoglobin (SFH) is advocated as an oxygen-transporting resuscitation solution. Hemoglobin has been shown to enhance endotoxin lethality when given intraperitoneally. It is possible that SFH could interact with endotoxin when used as an oxygen-transporting resuscitation system for trauma victims with contaminating wounds. To assess the effects of these two agents when given intravascularly, rabbits were infused with SFH (1.75 gm/kg) or albumin (controls; 1.75 gm/kg) with and without endotoxin. Two doses of endotoxin were used. At 14.5 ng/kg of Salmonella enteritidis endotoxin, no effect was seen in the albumin group. However, 50% of the hemoglobin group died. At 14.5 micrograms/kg, the albumin group showed hematologic alterations, but all animals lived. All SFH-treated animals died at the higher endotoxin dose. SFH alone caused cardiac abnormalities (bradycardia in 100%, sinus arrhythmias in 30%, and ventricular arrhythmias in 20%), liver abnormalities (necrosis in 40% and 240% increase in alanine aminotransferase activity by 6 hours), and intravascular thrombi (30%). The only hemoglobin-induced abnormality that was more frequent in the presence of endotoxin was ventricular arrhythmias (up to 75% of animals). Thrombin times were approximately 20% larger in all SFH groups compared with the albumin groups. By 6 hours after infusion, endotoxin prolonged the thrombin time even further, despite the lack of fibrinogen consumption. This study shows that endotoxin and SFH exert synergistic toxicity when SFH is given in a clinically relevant dose for an oxygen-transporting resuscitation system. Only minute quantities of endotoxin are needed to produce this phenomenon. We hypothesize that this synergism is endotoxin enhancement of hemoglobin toxicity.

Fluid resuscitation after an otherwise fatal hemorrhage: II. Colloid solutions.

We developed a fixed-volume porcine hemorrhage model that simulates the rapid exsanguination of combat or civilian trauma victims. In this study we compared the ability of colloid resuscitation solutions to prevent death after an otherwise lethal hemorrhage in 100 swine. The shed blood was replaced in a 1:1 ratio with either autologous whole blood (WB), untyped swine fresh frozen plasma (FFP), typed FFP, 5% human serum albumin (ALB), or normal saline (NS). Survival rate analysis indicated that WB was significantly better than FFP (untyped), ALB, or NS but not better than typed FFP. The 24-hour survival rates were: WB = 90%, typed FFP = 79%, untyped FFP = 56%, ALB = 57%, and NS = 25%. All deaths in the untyped FFP group suddenly occurred during or within 15 minutes after treatment in a recovering animal. Deaths in the ALB group steadily occurred for up to 2 1/2 hours after treatment. Analysis of hemodynamic, arterial blood gas, and acid-base data indicated that WB and FFP provided a better acid-buffering capacity in surviving animals than NS or ALB. We conclude that compatible FFP is a better resuscitation agent than ALB after an otherwise fatal hemorrhage because FFP is a better acid buffer.

The buffering capacity of crystalloid and colloid resuscitation solutions.

The buffering capacities of common colloid and crystalloid resuscitation solutions were compared in vitro. An equal volume of each resuscitation solution was titrated above and below its initial pH with 0.14 N sodium hydroxide or 0.11 N hydrochloric acid. The volume (+/- S.D.) of titration solution necessary to lower the pH one unit (7.1-6.1) in these solutions was less than 0.5 ml for normal saline, less than 0.5 ml for Ringer's lactate, 1.9 +/- 0.1 ml for Plasmalyte-A, 2.0 +/- 0.23 ml for Plasmalyte-R, 8.8 +/- 0.17 ml for human serum albumin (HSA), 45 +/- 2.2 ml for human fresh frozen plasma (FFP), and 50 +/- 6.6 ml for swine FFP. With the method of this in vitro study, human fresh frozen plasma was 25-50 times better as an acid buffer than the crystalloid solutions and approx. 5 times better than human serum albumin (HSA). On an equal volume basis, it was the superior resuscitation solution as a buffer, probably because of combined bicarbonate and protein content.

Characteristics of lympho-myelopoietic stem cells isolated from canine peripheral blood.

If hematopoietic stem cells (HSC) could be separated from peripheral blood, it might be possible to harvest these stem cells for potential clinical use. By leukapheresis techniques, we harvested mononuclear cells (MNC) from peripheral blood and then placed these cells over discontinuous stractan gradients of three densities (1.077 gm/ml, 1.071 gm/ml and 1.066 gm/ml). These separated cells were submitted to colony culture to identify colony-forming-unit activity for granulocyte-macrophage (CFU-C) and T-cell lymphocyte (CFU-L) cell lines. The lightest cells (1.066) contained most of the CFU-C and no CFU-L activity. Heavier cells (greater than 1.071) contained CFU-L and very little CFU-C activity. CFU-L colonies could be distinguished from CFU-C by their density and distinct morphological appearance. In addition, the amount of CFU-C could be increased in the animal by increasing the amount of blood processed (from 3.9 +/- .76 CFU-C/10(6) MNC to 6.7 +/- .35 CFU-C/10(6) MNC). This resulted in an increase of CFU-C collected from 7.6 +/- 2.1 CFU-C/10(6) MNC after the first equivalent blood volume to 22.5 +/- 3.4 CFU-C/10(6) MNC after the third equivalent blood volume processed. These results suggest that leukapheresis and gradient density separation may be useful procedures to obtain HSC.

Glycerol-glucose cryopreservation of platelets. In vivo and in vitro observations.

Extended storage of platelets can be achieved by cryopreservation. However, most cryopreservation techniques require extensive manipulation prior to administration, limiting their practicality. A simple cryopreservative system using glycerol and glucose as cryoprotectants would eliminate the need to wash the platelets after freezing, since neither of these agents is toxic. We evaluated such a system in vivo and compared the results to 72-hour liquid-stored platelets. The percentage of in vivo recovery was significantly less (p less than 0.01) for cryopreserved (21.1 +/- 3.4% [chi +/- 1 SD]) than liquid-stored (43.8 +/- 7.4%) platelets, but those frozen-thawed cells that were viable had normal survivals (8.4 +/- 1.7 days). Liquid-stored cell appeared to be less viable (5.9 +/- 1.8 days). These results indicate that cryopreservation with the glycerol-glucose system produces significant injury to the majority of platelets and therefore, is inadequate for general blood bank use.

Granulocyte progenitor cell (CFUC) harvest by continuous apheresis in dogs. Effects of blood volume and lithium on yields.

The ability to harvest large amounts of hematopoietic stem cells from blood would eliminate the more difficult approach of bone marrow harvest. Unfortunately, concentration of stem cells in the blood compartment is less than 1% of their concentration in bone marrow. Attempts to increase harvest of blood stem cells, as assayed by granulocyte progenitor cells (CFUC), have been only partially successful. Our study confirms previous reports that CFUC can be mobilized into the blood compartment in dogs, but this mobilization is rate-limited. Unlike platelets and granulocytes that are effectively harvested during the first blood volume processed by continuous apheresis, effective CFUC harvest begins during the second blood volume (606 +/- 97.9 CFUC/ml), peaks by the third (740 +/- 30 CFUC/ml), and remains constant through five blood volumes processed (700 +/- 272 CFUC/ml). Since blood CFUC concentration falls at the end of five blood volumes processed (40% of initial values), further continuous apheresis would not be effective. Treatment of animals with lithium did not improve CFUC harvest. These results show that apheresis procedures can be developed to a limited extent to increase the harvest of hematopoietic progenitor and stem cells.

Modification of hemoglobin - tetrameric stabilization.

Advantage has been taken of the unique affinity of DBBF for hemoglobin to stabilize the T state and crosslink tetrameric hemoglobin. The oxygen affinity has been further decreased by using PLP to produce a chemically unique molecule with a P50 of 32 under physiologic conditions. Furthermore, the modification is specific, requires only reagents that are commercially available or easily synthesized, and can be prepared in large quantities with up to 80% yield. The unique modified hemoglobin was purified by HPLC and the crosslink was found between the beta chains. This derivative, pyridoxalated-fumarate hemoglobin, sustained life in five rats that were 95% exchanged-transfused with the solution. Preliminary in vivo tests support this derivative as a desirable oxygen-transporting resuscitation fluid.

An in vivo comparison of CPD and CPDA-2 preserved platelet concentrates after an 8-hour preprocess hold of whole blood.

To see if citrate-phosphate-dextrose-adenine-two (CPDA-2) anticoagulant-preservative had an effect on the viability of platelets, we studied autologous in vivo recovery and survival in humans for platelet concentrates prepared from six units of blood drawn into CPDA-2 and compared them to six units drawn into citrate-phosphate-dextrose (CPD). These units were prepared from whole blood held at room temperature for 8 hours after collection and were then stored for 3 days at 22 +/- 2 degrees C. The recovery for platelets preserved in CPD was 39.0 +/0 4.8 percent and for platelets preserved in CPDA-2, 32.5 +/- 4.4 percent. The difference was not significant (p greater than 0.10). In order to estimate population differences, in vitro effects on in vivo viability were also evaluated. Six in vitro variables were studied but only pH at 72 hours (r = 0.77), platelet count (r = 0.64), and morphology score (r = 0.66) correlated to recovery. Only pH at 72 hours significantly influenced recovery (p = 0.007). By adjusting for individual pH differences, mean recovery for platelets stored in CPD was 37.5 percent, and for platelets stored in CPDA-2, 34.0 percent. The mean lifespan was 6.7 +/- 0.7 days for platelets preserved in CPD and 6.1 +/- 1.0 days for those preserved in CPDA-2. Although hemostatic function was not studied, these data support in vitro observations that platelets preserved with CPDA-2 are not different from platelets preserved with CPD, even after 8-hours of storage of whole blood at room temperature prior to platelet concentrate preparation.

Effect of delayed refrigeration on plasma factors in whole blood collected in CPDA-2.

Extension of the time within which whole blood may be separated into components offers logistic advantages for the operation of remote mobile drawing teams. We evaluated the effect of an 8-hour hold of whole blood at room temperature before preparation of components. Plasma coagulation activity and opsonic factor content were studied in 14 units drawn into the anticoagulant-preservative solution citrate-phosphate-dextrose-adenine (CPDA-2). At the time of collection, an additional 7-ml aliquot was drawn into 1 ml of CPDA-2, the plasma separated and frozen immediately. Components were prepared from whole blood units allowed to rest undisturbed at 22 +/- 1 degrees C for 8 hours. After 8 hours, a significant decrement of about 10 percent was found in the concentration of fibrinogen, plasminogen, fibronectin, and activity of Factor V. Factor VIII activities (VIIIAHF and VIIIAGN) were not significantly different after 8 hours. Our results indicate that room temperature storage for 8 hours before component processing has minimal effects on potentially labile plasma protein factors using CPDA-2 anticoagulant-preservative solution.

Canadian Red Cross lecture. Current concepts of oxygen-transporting blood substitutes.

Blood substitutes are being developed that will provide oxygen-transporting capabilities as well as volume replacement. Perfluorochemical and hemoglobin solutions have potential clinical use. A perfluorochemical blood substitute, Fluosol-DA 20%, is being used in clinical trials in several countries. These blood substitutes are not capable of totally replacing the need for blood transfusions but could be used temporarily in situations where blood is contraindicated or not available. They may be useful for a wide range of clinical conditions other than blood replacement, such as impending tissue ischemia. Before large-scale clinical use of these products is realized, more information is needed about drug efficacy and safety so that intelligent decisions can be made about indications for this type of transfusion therapy.

Glycoprotein changes in fresh vs. room temperature-stored platelets and their buoyant density cohorts.

Membranes from platelets obtained from normal human volunteers were isolated for evaluation of their glycoproteins. Values were measured in fresh and stored platelet concentrates at 72 and 96 hr (22 degrees +/- 2 degrees C). Polyacrylamide gels were used to separate the membrane glycoproteins. These were identified as GPI, GPII, GPIII, GPIV, GP77, and GP44. The relative amount of GPI (155,000 daltons) was about 30% less after 72 hr storage than in fresh platelets regardless of the change in the pH of the platelet concentrate. At 96 hr, only an additional 5% loss was seen. The 72 hr value for soluble glycoprotein, glycocalicin, was only 64% of the initial value. GP77 (77,000 daltons) and GP44 (44,000 daltons) became apparent or more prominent with storage. A correlation with pH of the platelet concentrate could be demonstrated for GPI but only with those units in which the pH rose during storage. Density-separated populations of fresh and stored platelets also were studied. They were separated on arabinogalactan (Stractan II) gradients for comparative studies of membrane proteins that might be affected, since platelets become less dense with storage. There was an equal loss of GPI in all populations. However, the change was most striking in the least dense (lightest) fraction because these cells started with 20% less GPI when fresh than did heavier cells. The smallest glycoprotein, GP44, was always present in the lightest platelets after storage, whereas only 40% of the concentrates showed GP77, in small amounts, associated with heavy platelets. Alterations in the membranes of platelets stored as platelet concentrates could result in their functional impairment and loss viability.

Buoyant density of platelets stored at room temperature as platelet concentrates.

Separation of platelets by buoyant density centrifugation was periodically performed on platelet concentrates stored up to 96 hr at room temperature. By 72 hr, platelets were much lighter, depending on pH, platelet concentration, and volume of the bag. The mean proportion of platelets in the light fraction (fraction 1) shifted from 4.3% when the concentrate was fresh to 52.2% at 72 hr and 53.6% at 96 hr. The majority of platelets had densities that ranged from 1.034 to 1.088 gm/ml after storage, whereas densities ranged from 1.054 to 1.088 gm/ml in fresh cells. With storage, the light cells became larger than when they were fresh and were mostly balloon-shaped; the heavy cells became smaller but retained their normal shape. Regression analysis showed that density distribution was highly correlated to pH. Most of the changes occurred after 12 hr; those changes that occurred during the initial 12 hr were not related to pH of the platelet concentrate. The changes were related to storage conditions and may reflect injury to the cells. The use of buoyant density separation may be a useful tool to study storage mechanisms and provide a means of separating cells modified by storage stress.

Adenine in blood preservation.

The recent Food and Drug Administration (U.S.) approval of a new blood preservative (CPDA-1) which contains adenine not only introduces a new blood product into the American blood banking system, but also heralds the advent of novel approaches to blood product preservation. The use of adenine to effect maintenance of red cell adenosine triphosphate (ATP), and hence to prolong storability, has a well-founded biochemical rationale. Effects of adenine on red cell metabolism are generally well understood, but effects on other blood components have not been fully delineated. The efficacy of adenine preservatives in enhancing the duration of red cell storage appears to outweigh the small risk of toxicity from free adenine. Clinical use of millions of units of adenine-preserved blood in Europe during more than a decade has resulted in only one report of possible adenine toxicity. Marginal acceptability of 24-hr 51Cr red cell recovery of packed red cells stored for 35 days in CPDA-1 has stimulated development and evaluation of an improved preservative (CPDA-2) which may extend blood storability beyond 35 days. A heightened awareness of the hematological consequences of prolonged storage has come with the extension of blood storage beyond 21 days. The concepts of component-specific preservation systems and optimal preservation systems have emerged as a result of experimentation on adenine preservatives. While the influence of adenine preservatives on American blood banking is yet to become manifest, the ultimte impact of adenine on blood preservation may be the development of novel systems which optimally preserve specific blood components at the option of the user.

In vitro evaluation of platelets stored in CDP-adenine formulations.

Little information is available about the effect of adenine and added glucose on stored platelets. Two new formulations, CPDA-2 and CPDA-3, contain 34 mg adenine per 63 ml preservative and extra glucose (1.75 and 2.0 times the glucose in standard CPD). We have studied the in vitro integrity of platelet concentrates stored in CPD, CPDA-1, CPDA-2, and CPDA-3 at 22 C for 72 hours. Morphology score, pH, platelet size, population distribution parameters, and electron microscopic ultrastructure did not show any adverse effects which could be ascribed to the presence of adenine or extra glucose or both. No differences in platelet adenosine triphosphate (ATP) concentration or plasma glucose utilization during storage were found between CPD and CPDA-1 platelets. The results suggest that adenine and added glucose in these preservatives are not detrimental to platelets in vitro by the measures employed.