Claes F. Högman

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.

White cells protect donor blood against bacterial contamination

The possible beneficial role of white cells (WBCs) in donor blood has been investigated with respect to their capacity to remove bacteria. Preparations of buffy coat and whole blood, containing as well as reduced of WBCs, were inoculated with Staphylococcus epidermidis, S. aureus, Escherichia coli, Pseudomonas aeruginosa, and Propionibacterium species. Upon storage at room temperature, the presence of WBCs resulted in a reduction of the bacterial content. Units inoculated with S. epidermidis and E. coli were completely cleared of bacteria within 5 to 24 hours. On the other hand, S. aureus, after an initial reduction in number, started to multiply. In WBC‐reduced units, the initial bacterial content remained unchanged for 5 hours, but the bacteria then exhibited vigorous growth within 48 hours in buffy coat and slower growth in whole blood. Propionibacterium sp. did not grow with or without WBCs. P. aeruginosa did not grow in buffy coat but showed a growth pattern similar to that of S. aureus in whole blood. The presence of WBCs in the donor blood during the first hours after collection thus seems to rid the blood of at least some species of bacteria. These results indicate that it would be favorable not to perform WBC reduction during blood collection and that several hours of contact can be needed to obtain sterility.

Platelet Concentrates in an Additive Solution Prepared from Pooled Buffy Coats.

Abstract. A new method for the preparation of platelet concentrates (PCs) is described. The source material is buffy coat (BC), prepared after keeping standard CPD whole‐blood units at room temperature for 6–12 h, followed by centrifugation at 3,500 rpm for 10 min (first series) or 4,000 rpm for 12.5 min (second series). BC, separated from plasma and red cells, was kept at room temperature for a further 8–12 h without agitation. Pools of 6 (first series) and 4 (second series) BCs were prepared using a sterile docking device and suspended in a platelet‐additive solution (PAS) containing sodium/potassium chloride, citrate, phosphate, and mannitol. After gentle centrifugation, the platelet‐rich supernatant was expressed to and stored in one (first series) or two (second series) 1‐liter polyolefine (PL‐732) containers. In the first series, the total number of platelets was 316 ± 59 times 109 per PC (yield 65%). However, when the method was applied at a routine scale, the yield varied considerably and was shown to be strongly dependent on the hematocrit of the BCs. A number of steps were taken to standardize the technique which resulted in an improved yield (77.3 ± 8.7%) with 316 ± 52 times 109 platelets (mean ±SD, range 203–490, n = 134), obtained from 4 BC pools and lower leukocyte contamination than before, 18 ±17 times 106 per preparation (range 1–73, microscopic counting, n = 38). The storage medium consisted of a mixture of plasma and PAS. Standardization of the hematocrit and volume of the BCs is essential, both for improvement of the yield and for providing a sufficient amount of glucose and bicarbonate to the platelets throughout storage. The method is particularly suitable in combination with automated techniques for blood component preparation which include BC removal. Blood gases, pH and adenosine triphosphate were maintained at satisfactory levels; the release of platelet factor 4 and lactate dehydrogenase was not different from traditional PC preparation and storage. However, the content of glucose was exhausted in many PC units towards the end of a storage of 6 days. When whole blood and BC are stored for such a long period, and when the gas permeability of their containers is poor, it is important, in order to avoid early loss of swirling, that the surface‐to‐volume ratio and the gas permeability of the PC containers is sufficiently large to allow good gas exchange.

Photochemical inactivation of bacteria and HIV in buffy-coat-derived platelet concentrates under conditions that preserve in vitro platelet function

Background and Objectives: A photochemical process has been tested for the inactivation of viruses and bacteria in buffy‐coat derived platelet concentrates (BC PCs). Materials and Methods: BC PCs in 35% CPD plasma and 65% platelet‐additive solution (PAS III) were exposed to photochemical treatment (PCT) with 150 μM of the psoralen S‐59 and a 3 J/cm2 treatment with longwavelength ultraviolet light (UVA, 320–400 nm). Platelet function was evaluated following PCT using a panel of in vitro assays. Results: This PCT process was highly effective at inactivating gram‐positive bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis) and gram‐negative bacteria (Enterobacter aerogenes, Pseudomonas aeruginosa, Serratia marcescens). No viable bacteria were detected following PCT and 7 days of platelet storage while bacterial growth was detected in paired untreated control BC PCs. Complete inactivation of the gram‐positive Bacillus cereus was achieved only in one of two replicate experiments with BC PCs. PCT was also highly effective for inactivation of human immunodeficiency virus HIV‐1 in BC PCs inoculated with approximately 106 tissue culture infectious doses per milliliter (TCID5n/ml) of cell‐associated HIV‐1. Rapid inactivation was observed with increasing UVA doses: with 150 μM S‐59 and a 1 J/cm2 treatment of UVA, a reduction of 5:6±0.5 log TCID50/ml was achieved, and a reduction of >6.4 log TCID50/ml was achieved with 150 μM S‐59 and a 3 J/cm2 treatment of UVA. No physiologically relevant differences in platelet functions were found between the test and the control BC PCs during 7 days of storage. Conclusion: PCT with 150 μM S‐59 and a 3 J/cm2 UVA treatment does not adversely affect in vitro properties of BC PCs stored at 22°C for 7 days. The PCT process inactivated bacteria and HIV‐1 inoculated into the BC PCs. These results extend the earlier reported efficacy of PCT apheresis PCs to BC PCs.

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.

Platelet concentrates in an additive solution prepared from pooled buffy coats : in vivo studies

Leukocyte‐depleted platelet concentrates were prepared from pools of 4 buffy coats on the day after blood collection (BC‐PC). The storage medium was composed of citrate phosphate dextrose plasma and a platelet‐additive solution. Autologous transfusions of 111In‐labelled platelets in 9 healthy volunteers were performed on the day of preparation (day 1) and on day 5. The recovery was 54.6±8.7 (day 1) and 51.9±10.4% (day 5), T1/2 was 101±28 and 61±9 h, respectively. The survival was 8.3±1.7 and 5.7±1.0 days, respectively, using linear plot, and 7.8±2.0 and 5.8±0.5 days using the multiple hit method. In a prospective clinical study a comparison of the corrected posttransfusion increments was made between BC‐PCs and apheresis‐PCs, and between fresh (1–2 days) and stored (3–5 days) preparations. No difference was found between BC‐PCs and apheresis PCs. However, fresh BC‐PCs gave higher increments than stored BC‐PCs. A slight numerical difference between fresh and stored apheresis‐PCs was not statistically significant. It is concluded that the BC‐PC method results in platelets of equal quality to apheresis‐PC.

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.