Relationships between in Vivo Survival and (1) Density Distribution, (2) Osmotic Fragility of Previously Frozen, Autologous, Agglomerated, Deglycerolized Erythrocytes

In 39 autotransfusions, significant correlations were observed between (1) density distribution and (2) osmotic fragility and 24-hour posttransfusion survival of chromium-labeled human erythrocytes preserved with glycerol using a slow-freeze agglomeration technic.
The temperature of storage and the volume of isotonic saline used to disaggregate the agglomerated red cell mass were critical; optimum results were observed following storage of the preserved cells at –80 C and with disaggregation of the agglomerated red cell mass of each unit with 250 cc of isotonic saline.
These data indicate that preserved cells with a marked increase in density and a marked decrease in osmotic fragility had a decreased posttransfusion survival. Within 24 hours following the infusion of preserved cells characterized by a marked increase in density and a marked decrease in osmotic fragility, temporary sequestration of a portion of cells was observed, with release of these cells noted on the third to fourth day following the transfusion.     

Analysis of Erythrocyte Survival Curves Obtained Simultaneously by 51Cr and an Automated Differential Agglutination Technic

Using both a radioactive chromium and an automated differential agglutination (ADA) technic donor erythrocyte survivals were measured in 19 subjects. The data were analyzed to determine the presence of age‐dependent (linear) and random (exponential) destruction. The erythrocyte lifespan was calculated from the rate of linear destruction. In all cases it was possible to estimate the erythrocyte lifespan with the ADA technic. Significant random destruction of red blood cells was observed in nine patients. With the ⁵¹chromium technic it was possible to estimate the erythrocyte lifespan in only ten cases; these values were similar to those obtained with the ADA technic. It was not possible to determine the erythrocyte lifespan in the remaining nine cases; the best estimates were too large. Our inability to measure erythrocyte lifespan satisfactorily with the ⁵¹chromium technic may have been related to the preferential labeling of young red blood cells in vitro .     

Automated Differential Agglutination Technic to Measure Red Cell Survival I. Methodology

Experiments were performed to prove the validity of an automated differential hemagglutination test to measure the survival of transfused red cells.
Four antibodies, anti-A, anti-B, anti-CD, and anti-M, were used to perform differential hemagglutination. This method permitted measurement of the survival of at least two compatible but antigenically identifiable red cell populations in the same recipient.
The measurements obtained with the automated method were less variable than those obtained simultaneously with a manual method.     

Therapeutic Effectiveness of Homologous Erythrocyte Transfusions Following Frozen Storage at ‐80 C for up to Seven Years

Cohn‐processed red blood cells that had been stored for as long as seven years at ‐80 C., washed by the ADL procedure and then stored at 4 C for up to 48 hours, showed approximately 90 per cent 24‐hour recovery in vivo by an automated differential agglutination (ADA) technic, recovery in vitro of approximately 90 per cent, and an index of therapeutic effectiveness of approximately 80 per cent. Washing Huggins‐preserved red blood cells with EDTA by the Huggins process produced a significant deterioration (decreased 24‐hour posttransfusion survival and decreased recovery in vitro ) following storage at ‐80 C for as long as three years. In two of seven patients studied the Huggins‐processed red blood cells that had been stored at ‐80 C for 1.8 years and longer and washed by the Huggins procedure showed intravascular destruction of the compatible nonviable red blood cells. Huggins‐preserved red blood cells with EDTA that had been stored at ‐80 C up to 1.6 years showed, following washing with an electrolyte solution in the ADL bowl, a somewhat better 24‐hour ADA survival, better recovery of the preserved red blood cells, lower supernatant hemoglobin concentrations, and higher intracellular potassium levels on the day of washing and resuspension. These findings suggest that Hugginspreserved red blood cells following storage at ‐80 C for one and one half years or more should not be washed by the Huggins dilution/agglomeration procedure.     

Oxygen consumption, platelet aggregation and release reactions in platelets freeze-preserved with dimethylsulfoxide

Platelets were frozen with 4% or 5% DMSO at an overall rate of 2 to 3 C per minute and were stored at −80 C for as long as 10 months. They were washed with DMSO-plasma and acid-citrate-dextrose (ACD) solutions and were stored in 30 ml of autologous plasma at room temperature for about three hours before transfusion. Measurements were made of oxygen consumption, platelet aggregation and release reaction, platelet factor- 3 and-4 activities, and platelet response to hypotonic stress. Platelet basal and latex stimulated oxygen consumption were found to be significantly impaired; platelet aggregation response to ADP, epinephrine, and collagen were decreased; platelet ATP and ADP content and release reactions were decreased; platelet antiheparin activity (platelet factor-4 level) was decreased; and the platelet response to hypotonic stress was impaired. What the results of these in vitro tests mean in relation to in vivo survival and hemostatic function of preserved platelets was not established.     

Clinico-pathological Correlations and Long-term Follow-up of 253 United Kingdom Patients with IgA Nephropathy. A report from the MRC Glomerulonephritis Registry

The Medical Research Council's Glomerulonephritis Registry wasused to study clinicopathological correlations and renal survivalin patients with IgA nephropathy reported between 1978 and 1985.IgA nephropathy was the histological diagnosis in 9.3 per centof all renal biopsies reported to the registry during this period,and in 18.1 per cent of those with a primary glomerulonephritis.The 10-year cumulative renal survival rate accounting for censoreddata (Kaplan-Meier) was 83.3 per cent. Univariate analysis ofsurvival curves (log-rank test) found the following parametersto be significantly correlated with poor renal survival: serumcreatinine >120 µmol/l (p<0.001), hypertension(p<0.001), serum albumin <40 g/l (p<0.005), proteinuria>1 g (p<0.025), age >30 years (p<0.025), and focalmesangial proliferation (p<0.05). There was no significantdifference in renal survival between males and females. Multivariateanalysis (Cox's proportional hazards model) revealed that onlya serum creatinine of > 120 µmol/l and a serum albuminof <40 g/l were independently predictive of outcome. These findings indicate marked similarities between the UK experienceof IgA nephropathy and the published European experience. IgAnephropathy is not a benign condition in the UK and patientswith impaired renal function and/or those with a reduced serumalbumin are significiantly more likely to progress to end-stagerenal failure within 10 years.     

Observations on Autologous, Previously Frozen, Deglycerolized, Agglomerated, Resuspended Red Cells

Forty-four units of human erythrocytes preserved with glycerol, the slow-freeze technic, and agglomeration were evaluated by autotransfusions of chromium-labeled 10 cc aliquots. The shipment of frozen glycerolized red cells in dry ice and of thawed, deglycerolized, resuspended red cells in wet ice did not adversely affect the in vivo or in vitro measurements. Excessive in vitro loss of cellular hemoglobin and unacceptable posttransfusion survival were observed when preserved erythrocytes were stored at + 4 C for longer than 24 hours, at —20 C for longer than three days, and at — 30 C for longer than seven days between periods of storage at — 80 C. A storage period at —20 C alone for 50 days resulted in poor preservation.
Adenine supplementation of the glycerolized red cells (0.6 mM per liter) prior to freezing did not change significantly the in vivo or in vitro characteristics of red cells stored at the warmer temperatures.
Highly significant correlations were noted between the 24-hour posttransfusion survival and MCV, MCHC, osmotic fragility, and density distribution of the preserved red cells.     

Viability of Glycerolized Red Blood Cells Frozen in Liquid Nitrogen

Freeze-preservation of human red blood cells with a low concentration of glycerol (approximately 20 per cent w/v), liquid-nitrogen refrigeration, and a stainless steel container was evaluated by measurements of the posttransfusion survival of 10-ml samples of ⁵¹chromium-labeled autologous red blood cells, together with several measurements in vitro. Removal of glycerol prior to transfusion (the major technologic problem) was carried out both by serial centrifugation (batch washing) and by continuous centrifugation, using either predilution or sequence wash cycles. A biologic product of glycerolized red blood cells was prepared to the following specifications: the recovery in vitro of at least 90 per cent of the red blood cells after thawing and washing was achieved, together with 24-hour posttransfusion survival in vivo of approximately 85 per cent, a total amount of supernatant hemoglobin in the unit on the day of washing of approximately 450 mg, a preparation time of approximately 30 minutes, and a total volume of wash solution of less than 3.0 liters. The present urgent need in frozen blood preservation is for systems which use disposable software in a completely automated wash cycle.     

The survival, function, and hemolysis of human RBCs stored at 4 degrees C in additive solution (AS-1, AS-3, or AS-5) for 42 days and then biochemically modified, frozen, thawed, washed, and stored at 4 degrees C in sodium chloride and glucose solution for 24 hours

BACKGROUND: A study was done to assess the quality of RBCs stored at 4 degrees C in AS-1, AS-3, or AS-5 for 42 days before biochemical modification and freezing. STUDY DESIGN AND METHODS: RBCs were stored at 4 degrees C for 42 days in AS-1, AS-3, or AS-5 and then biochemically modified with pyruvate, inosine, phosphate, and adenine solution (Rejuvesol), frozen with 40-percent (wt/vol) glycerol, and stored at -80 degrees C for at least 2 months. The RBCs were deglycerolized by the use of a cell washer (Haemonetics 115), and stored for 24 hours at 4 degrees C in a 0.9-percent sodium chloride and 0.2-percent glucose solution before the autologous transfusion. RESULTS: The mean freeze-thaw-wash recovery process produced RBC recovery values of 85 percent, with the mean 24-hour posttransfusion survival at 75 percent, and the mean index of therapeutic effectiveness at 64 percent for the RBCs stored at 4 degrees C in AS-1, AS-3, or AS-5 for 42 days before biochemical modification and freezing. All the units exhibited normal or slightly higher than normal 2,3 DPG levels after deglycerolization and postwash storage at 4 degrees C for 24 hours. CONCLUSION: RBCs stored in AS-1, AS-3, or AS-5 at 4 degrees C for 42 days and then biochemically modified with pyruvate, inosine, phosphate, and adenine and glycerolized, frozen, washed, and stored at 4 degrees C for 24 hours before autologous transfusion had acceptable in vitro and in vivo measurements.