Kenneth Hedlund

The Bottom and Top System: A New Technique for Blood Component Preparation and Storage

Abstract. A new, automated technique for the preparation of blood components is described. A system of 3 or 4 integrally connected plastic containers (Optipac®) is handled by a new type of extractor (Optipress®). The container in which the blood is collected has an outlet at the top and another at the bottom. After normal centrifugation to obtain separation of the blood components, these are squeezed out from the top and bottom simultaneously under control of a photocell. The primary separation step results in three components: a leukocyte‐poor red‐cell suspension in SAGM medium, CPD plasma, and a buffy‐coat preparation. The system has been tested in two laboratories (lab A and lab B). A ‘heavy‐spin’ centrifugation to obtain a maximum yield of cell‐poor plasma gave the best removal of leukocytes from the red cells; the remaining leukocyte content was 0.46 ± 0.25 (lab A) and 0.5 ± 0.4 (lab B)x 109/red‐cell unit. Platelet concentrates can be prepared either the normal way via platelet‐rich plasma or from buffy coat. Red‐cell 24‐hour autologous posttransfusion survival using labeling with 51Cr was 87.5 ± 4.1% (lab A) after 35 days, and 84.2 ± 4.2% (lab A) and 77.5 ± 1.5% (lab B) after 42 days. Red‐cell morphology and fluidity compared favorably to previous studies using the same additive solution in traditional plastic‐bag systems. The total adenine nucleotide concentration was maintained normal for 42 days. Storage hemolysis after 6 weeks was slightly higher after a heavy spin, 0.52 ± 0.19% (lab A) and 0.23 ± 0.10% (lab B), than after a light spin, 0.31 ± 0.15 and 0.20 ± 0.09%, respectively. The formation of fibrinopeptide A was very low during the first 2 weeks and then increased to 15 ± 15 nmol/1 after a heavy spin and to 33 ± 11 nmol/1 after a light spin. Kallikrein and spontaneous proteolytic activity increased from day 14 on in the light‐spun units but not in the heavy‐spun ones. Clinical studies were made in two hospitals. A total of 1,492 transfused blood components prepared with the new system were compared with 1,169 components prepared with a traditional four‐container system for making buffy‐coat poor red‐cell suspensions. No unexpected transfusion reactions occurred. The frequency of febrile reactions was 3/516 (new method) and 2/466 (controls) in a group of patients with predominantly myeloproliferative diseases. The system appears to be a significant improvement for automated preparation of high‐quality blood components.

Clinical usefulness of red cells preserved in protein-poor mediums.

Blood is normally collected into a combined anticoagulating and preserving medium. We performed a study to ascertain whether improvements could be made by separation of these two functions. Addition of saline-adenine-glucose solutions (40 to 100 ml per blood unit) to buffy-coat-poor red-cell concentrates allowed storage for as long as 35 days with 24-hour erythrocyte post-transfusion survival of 83 +/- 6.8 per cent (+/0 S.D.). Potassium leakage was lower, and in vitro hemolysis somewhat higher than that of whole blood. The microaggregate content after 21 days was 16 per cent of that in whole blood. In over-pressure transfusions the flow rate of red cells was the same with red-cell concentrates to which 80 to 100 ml of suspension medium had been added (hematocrit less than or equal to 60 per cent) as with whole blood. Removal of the buffy coat was essential to reduce hemolysis. We conclude that red cells can be successfully stored in a simple protein-poor medium.

Studies on the Mechanism of Human Red Cell Loss of Viability during Storage at +4 °C in vitro

Abstract. Red cells stored in SAGM medium for 42 days at +4°C were rejuvenated by bicarbonate, pyruvate and adenosine. Autologous 24‐hour posttransfusion survival was determined in untreated as well as rejuvenated cells and showed an improvement from 77.4±4.7 to 89.2±7.2%. The erythrocyte adenylate energy charge decreased relatively more than the total adenylate concentration during storage, but the latter correlated better with posttransfusion red cell survival. Considerable deteriorations in red cell morphology (expressed as morphology index) and in deformability (measured as red cell fluidity) were observed during storage but were partly reversed by rejuvenation. The morphology index and the posttransfusion survival showed a significant correlation (r = 0.95, p<0.005) after, but not before, rejuvenation, indicating that the remaining changes are more permanent and decisive of survival. It is suggested that, in the proportion of stored erythrocytes which respond to rejuvenation, the capacity and time dependence of recovery of normal shape and flexibility are important.

Red blood cell preservation in protein-poor media. I. Leukocyte enzymes as a cause of hemolysis.

Red blood cells were prepared from CPD whole blood concentrated to 90 per cent hematocrit, diluted with saline‐adenine‐glucose (SAG) media and then stored for 35 days. The dilution was undertaken to improve the flow properties of the blood and to provide the cells with glucose and adenine allowing prolonged storage. Several different compositions were tested. ATP could be maintained at about 70 per cent of the initial level and the 24 hour red blood cell posttransfusion survival was 82 per cent. The leakage of potassium was less than in CPD‐adenine whole blood if the dilution volume was half or less of the red blood cell volume. The only problem was that the hemolysis was greater in SAG‐stored blood than in CPD whole blood or undiluted CPD red blood cell concentrate. Hemolysis could be reduced by removal of huffy coat cells or by storage of blood in the presence of synthetic enzyme inhibitors. A chymotrypsin‐like enzyme isolated from human leukocytes had a potent hemolytic effect. The hypothesis is presented that red blood cells stored in an electrolyte medium in the presence of leukocytes undergo increased hemolysis because they lack the protecting effect of plasma enzyme inhibitors which normally inhibit any hemolytic enzymes leaking from damaged leukocytes. The study has practical implications for the prolonged storage of buffy‐poor red blood cell preparations to be used in transfusion therapy.

Red Cell Preservation in Protein‐Poor Media: III. Protection Against in vitro Hemolysis

Red cells stored under blood bank conditions normally show less than 1% spontaneous in vitro hemolysis even after 5 weeks; larger hemolysis may be found if the cells are suspended and stored in a saline‐adenine‐glucose (SAG) solution with very little trapped plasma. Delay of the addition of the suspension medium, return of 25 ml plasma after a maximal plasma harvest, addition of mannitol 10–30 mmol.1‐1 to the suspension medium were alternative and effective ways of keeping the spontaneous lysis within normal limits. Mechanical traumatization (centrifugation or shaking) caused considerably more damage to the red cells when these were highly concentrated than when they were diluted. A cell suspension in SAG is a more suitable product for hemotherapy than strongly packed red cell concentrates.

Red Cell Suspensions in SAGM Medium Further Experience of in vivo Survival of Red Cells, Clinical Usefulness and Plasma-Saving Effects

Abstract. Red cells depleted of buffy coat and more than 90% of the plasma were suspended and stored in a medium composed of sodium chloride, adenine, glucose and man‐nitol (SAGM). The 24‐hour posttransfusion survival of 51Cr‐labeled red cells was 83.5 ± 5.3% (n = 4) after storage for 35 days and 77.4 ± 4.7%o (n = 6) after 42 days. No abnormal in vivo hemolysis occurred as judged from posttransfusion haptoglobin consumption studies. No abnormal body temperature elevation was found at continuous pertrans‐fusion recordings. The frequency of febrile or urticaria1 transfusion reactions was 0.19% as compared to 0.68% during a whole‐blood transfusion period. Since a mean of 280 ml of plasma can be collected from each blood unit the plasma‐saving effects of the system are considerable. Favorable large‐scale clinical experience is reported.

Effects of Oxygen on Red Cells during Liquid Storage at +4°C

Red cells collected in CPD and suspended in SAGM medium were stored in plastic (PVC) containers for 42 days at +4°C. Comparison was made between aerobic storage (normal air exposure) and anaerobic storage (exposure to nitrogen gas). The air‐exposed units showed a strong increase in pO2 and oxygen saturation as a result of oxygen penetration into the bags from outside. This resulted in a decrease in ATP and adenylate energy charge, a slower metabolization of adenine and hypoxanthine to AMP and IMP, respectively, and a faster decrease in red cell fluidity. To explain the findings it is concluded that aerobic storage causes an increased need of high‐energy phosphate groups, possibly used for replacement of the phospholipid membrane bilayer or in repair of phosphate bonds in the cytoskeleton. It is further proposed that a slight formation of hydrogen peroxide from free oxygen radicals moderately increases the oxidation of reduced (GSH) to oxidized (GSSG) glutathione and slightly enhances the need for reduced nicotinamide‐adenine dinucleotides mainly provided by increased flux through the pentose phosphate shunt.

Storage of red cells in a CPD/SAGM system using teruflex® PVC

Abstract. A quadruple plastic bag system made of Teruflex® PVC (Terumo) was tested. Red cells separated from plasma and buffy coat were resuspended in SAGM solution. After storage for 42 days, the 24‐hour posttransfusion autologous survival was 73.3±6.6% (X̄ ± SD), range 61.8–80.7%, n = 10. The shape of the cells was changed so that normal or slightly abnormal morphology was found in 37±7% of the cells after 42 days. Spontaneous in vitro hemolysis was 0.63±0.32% (range 0.28–1.13). A sufficient glucose reserve still remained at the end of storage, but glucose consumption and lactate production were impaired during the last 2 weeks due to the increasing acidity. By extrapolation from other studies it is suggested that 35 days is a more suitable shelf life than 42 days in this system.