A method for the cryopreservation of red blood cells using hydroxyethyl starch as a cryoprotectant.

A simple, quick, inexpensive and long term method of cryopreserving human red cells using hydroxyethyl starch (HES) as a cryoprotectant is described. No sophisticated equipment or skilled labour is required. As HES is a plasma expander, it does not have to be removed prior to transfusion, thereby obviating the requirement for a washing stage. This allows 60 units/h to be prepared. As the microbiological integrity of the system is maintained, there is potential for prolonged storage post-thaw at 4 degrees C. Recoveries of 99% and 30 min saline stabilities in excess of 91% have been consistently achieved. P50O2 and 2,3DPG values are not significantly decreased.

The development of hemoglobin solutions as red cell substitutes.

It is clear from the trials described here that the number of different products being tested and the potential variation between batches of the same product present major problems in evaluating the safety and efficacy of hemoglobin-based oxygen carriers. The recent CBER "Points to Consider" document [42] makes clear that an understanding of the safety of oxygen carriers in humans is of paramount importance. In the event of phase II or indeed phase III trials being approved, the need may still remain for additional phase I or preclinical studies, particularly as unwanted or toxic properties of the solutions affect efficacy. It is likely that demonstrating safety and efficacy in acute hemorrhagic shock will be the most difficult task, as this is a complex clinical indication and is often accompanied by multisystem damage. The use of a hemoglobin-based oxygen carrier in this setting must have a distinct advantage over a plasma expander alone. In the application of perioperative transfusion, a decreased requirement for red cell transfusion has already been accepted as a basis for the efficacy for erythropoietin. However, in the case of a hemoglobin-based oxygen carrier, the reduction of red cell requirement in perioperative procedures would need to be balanced against any adverse drug reactions or unacceptable hemodynamic effects that may be caused by the product. It appears that there are still numerous hurdles to overcome in the development of hemoglobin-based red cell substitutes. Before these products can become established in medical practice, it is imperative that the potential mechanisms of toxicity of cell-free hemoglobin are clearly understood. Approval of hemoglobin-based oxygen carriers for clinical use will depend not only on clear demonstration of both safety and efficacy but also on risk-versus-benefit issues. Our understanding of the physiological effects of these products will evolve as progress is made in their clinical evaluation.

Tetanus antitoxin titres in military personnel.

A study was made of the titres of tetanus antitoxin in the sera of 186 recruits and 886 serving personnel. All were found to have titres of more than 0.01 IU per ml, the titre regarded as sufficient to provide protection from tetanus. The percentage of recruits with tetanus antitoxin titres less than 0.1 IU per ml was significantly greater than the percentage of serving personnel with antitoxin titres less than 0.1 IU per ml and the overall geometric mean titre of the antitoxin in the sera of the recruits was significantly lower than that in the sera of the serving personnel. In the light of the finding that all of the recruits had protective levels of tetanus antitoxin and in view of the imminent withdrawal of TABT Vaccine, alternative strategies for immunisation against tetanus are presented.

Xanthone additives for blood storage that maintain its potential for oxygen delivery. I. 2-Hydroxyethoxy- and 2-ethoxy-6-(5-tetrazoyl) xanthones in citrate-phosphate-dextrose-adenine (CPDA-1) blood.

Two xanthones, 2-hydroxyethoxy-6-(5-tetrazoyl) (BW A440C) and 2-ethoxy-6-(5-tetraozyl) (BW A827C), are members of a chemical series tested in vitro as potential additives to citrate-phosphate-dextrose-adenine (CPDA-1) medium for blood storage. P50 was maintained in the presence of these compounds during 42 days' storage by a partial maintenance of 2,3 diphosphoglycerate (2,3 DPG) and by a direct effect on hemoglobin previously reported for BW A827C. Red cell 2,3 DPG levels for BW A440C (n = 5), BW A827C (n = 5), and control (n = 6), respectively, were 3.38 +/- 0.47, 3.44 +/- 0.25, and 1.20 +/- 0.10 mM +/- SEM on day 7; 1.16 +/- 0.13, 1.52 +/- 0.37, and 0.16 +/- 0.02 mM on day 21; and 0.67 +/- 0.09, 0.61 +/- 0.08, and 0.06 +/- 0.006 mM on day 42. Red cell adenine triphosphate levels at the same time intervals were 1.84 +/- 0.09, 1.46 +/- 0.18, and 2.11 +/- 0.04 mM; 2.10 +/- 0.05, 2.07 +/- 0.17, and 2.13 +/- 0.05 mM; and 1.42 +/- 0.13, 1.37 +/- 0.13, and 1.38 +/- 0.06 mM, respectively. The degree of hemolysis was less with the addition of the compounds, and the methemoglobin formation, plasma Na+ and K+, and lactate production were unaffected by the compounds.

The effects of arginine, dextran and Haemaccel infusions on urinary albumin, beta 2-microglobulin and N-acetyl-beta-D-glucosaminidase.

Male volunteers were infused with L-arginine dextran and Haemaccel. Arginine (0.5 g/kg body weight infused over 30 min) resulted in transient highly significant increases in urinary albumin (p less than 0.001), beta 2-microglobulin (p less than 0.001) and N-acetyl-beta-D-glucosaminidase [NAG] (p less than 0.001). These effects lasted less than 120 min. Dextran 40 and 70 (500 ml infused over 2 h) did not affect urinary albumin, beta 2-microglobulin or NAG excretion. Haemaccel (8 ml/kg body weight infused over 2 h) resulted in significant increases in urinary albumin (p less than 0.05) and beta 2-microglobulin (p less than 0.01) during the second hour of the infusion. It also caused a biphasic increase in urinary NAG excretion, the initial peak (p less than 0.05) coinciding with the peak of albumin and beta 2-microglobulin excretion. The second peak which was more defined (p less than 0.01) occurred 21-24 h after the beginning of the infusion. Neither arginine or Haemaccel have been reported to be nephrotoxic whereas dextran infusions are a well recognised cause of acute tubular necrosis. These data indicate that increases in urinary beta 2-microglobulin and NAG are not always reliable indicators of nephrotoxicity or renal tubular cell damage.

A prospective trial of young red cells in 48 patients with transfusion-dependent thalassaemia.

Young red cells (YRBC) prepared on a cell washer were compared to whole blood in a randomized prospective trial in 48 transfusion dependent patients with thalassaemia major. A minor but statistically significant decrease in blood consumption was observed in the group receiving YRBC. However, no difference was seen in transfusion interval, mean haemoglobin and rate of haemoglobin fall between the two groups. Filtered blood was shown to be as effective as frozen blood in eliminating non-haemolytic febrile transfusion reactions in all the trial patients. We conclude that any reduction in the rate of iron loading brought about by the use of YRBC is clinically insignificant and does not justify the expense, time and work required to produce young red cells for use in a large transfusion dependent thalassaemic population.

Young red cell preparation--a comparison of available methods.

Four possible methods of preparing young red cells (YRBC) which exploit the relationship between red cell ageing and density are compared. Of these, two cell separators and the IBM cell washer are shown to be capable of producing YRBC suitable for clinical use. YRBC prepared from the IBM 2991 cell washer are recommended for clinical trials because of lower cost, greater convenience and lower white cell contamination compared to cell separator-derived YRBC.

Effects on renal tubular protein reabsorption of the infusion of free haemoglobin.

Hydroxy-ethyl-starch cryopreserved blood with a mean free haemoglobin content of 260 mumol/l (SD 57 mumol/l) was infused into five normal volunteers. The renal clearance of six proteins of differing molecular weight was measured together with an analysis of the qualitative changes in protein excretion during the infusion and over the following 48 h. Other aspects of renal tubular function were assessed by the measurement of urinary N-acetyl-beta-D-glucosaminidase, phosphate and amino acids. Increased clearance of low molecular weight proteins and N-acetyl-beta-D-glucosaminidase occurred during the 12 h following infusion. beta 2 microglobulin clearance X 10(3) creatinine clearance rose from a mean of 0.4 (SD 0.1) to a mean of 74.5 (SD 32.3) and N-acetyl-beta-D-glucosaminidase from 54.2 units/mmol creatinine (SD 18.0) to 1525 units/mmol creatinine (SD 2318). Increased amounts of low molecular weight proteins were detected in the urine by crossed immunoelectrophoresis. Creatinine clearance remained unaltered. It is argued that these changes may be caused by the competitive inhibition of low molecular weight protein reabsorption in the renal tubule by filtered haemoglobin.

Blood usage in transfusion-dependent patients. A theoretical model.

A model for blood requirements of transfusion-dependent patients is presented to relate the transfusion interval in days (I), the percentage fall in hemoglobin (F), and the time in days after a transfusion during which no red cell loss occurs (S). The model applies to both normal blood (S = 0) and to young red cell (YRBC) transfusions, giving I = (S + 120)/(200/F - 1) The model predicts prolongation of transfusion interval by use of YRBCs, and is also of use in establishing whether excess red cell destruction or significant red cell production is present in a transfusion-dependent patient receiving either normal blood or YRBCs.

A clinical study of low molecular weight-hydroxyethyl starch, a new plasma expander.

1. The pharmacokinetics of a new plasma expander, low molecular weight-hydroxyethyl starch (LMW-HES) were examined in six normovolaemic men. 2. One hour post-infusion, 13.5% of the total dose of LMW-HES injected was excreted in the urine, 50.2% was present in the intravascular space, and 36.3% was unaccounted for. 3. Twenty-four hours post-infusion, 65.5% of the total dose of injected LMW-HES had been excreted in the urine, 4.1% remained intravascularly, and 30.4% was unaccounted for. 4. The plasma volume increased rapidly from a mean value of 45.7 ml kg-1 to a maximum value of 57.9 ml kg-1 immediately post-injection, then gradually returned to normal over 24 h. 5. The infusion of an average of 58.1 g had no effect on ESR, renal and hepatic biochemical indices. 6. LMW-HES appears to be safe and effective, and should be of value clinically when rapid and short-lived augmentation of the plasma volume is required.

Transfusion of hydroxyethylated amylopectin-protected frozen blood in man. I. Plasma clearance and renal excretion of the cryoprotectant.

In man following the autologous transfusion of blood previously frozen with 14% low molecular weight-hydroxyethylated amylopectin (cryo-HES), the clearance of this material from the intravascular space was compound, and appeared to consist of exponential components. The overall half-life -- however, was 10.6 +/- 3.0 (SD) h. Approximately 17% of the total infused cryo-HES was excreted in the urine 1 h postinjection, and 40% by 72 h. The erythrocyte sedimentation rate (ESR) was not affected by the presence of this substance in the bloodstream of the recipient. The results indicate that cryo-HES is removed rapidly following the transfusion of blood previously frozen with this material.

Plasma clearance and renal excretion of erythrocyte cryoprotectant hydroxyethylated amylopectin.

The value of low molecular weight-hydroxyethylated amylopectin (cryo-HES) as an extracellular cryoprotectant has been demonstrated in vitro. It is important that details of the intravascular persistence and urinary excretion be determined to compare with data already available as other grades of HES and with data on transfusion of cryo-HES cryoprotected blood. Following a single 400 ml (14% solution) infusion in man, the intravascular clearance of cryo-HES was well described mathematically by the equation: y = 3.30+6.49e-0.15kappa. The plasma concentration of cryo-HES fell to half its peak value in approximately 9.6 h. Approximately 20% of the total infused cryo-HES was excreted in the urine during the first post-injection hour, and 50% by 72 h. The ESR was not altered significantly by the presence of this material. The present study indicates that cryo-HES is eliminated rapidly and may thus be safe for transfusion to recipients of frozen blood.

Sunlight and hypercalciuria.

Urinary calcium and magnesium excretion was measured in two groups of soldiers leaving the temperate climate of the united Kingdom for service in the Persian Gulf. In one group urinary calcium levels and magnesium/calcium ratios were similar, ten days after arrival in the Gulf during the "cold season", to those found in the U.K. The other group went to the Gulf in the "hot season", and calcium excretion rose immediately to levels comparable with those found in the first group after eight months. Mg/Ca ratios fell to levels seen in stonformers, and 2 of 91 soldiers followed up for three years have had urinary calculi. Increased exposure to sunlight seems to be the most likely cause of the hypercalciuria.