Prestorage removal of Yersinia enterocolitica from red cells with white cell-reduction filters.

Prestorage removal of phagocytic white cells (WBCs) may increase the survivability of contaminating bacteria in units of stored red cells. Fourteen units of whole blood were inoculated with 65 colony-forming units per mL of Yersinia enterocolitica (serotype O:3) and processed into AS-3-preserved red cells. Five red cell units were filtered with a prototype third-generation filter and five red cell units with a second generation filter. WBC reduction was performed on the day of collection. Four red cell units were not filtered. Three noninoculated whole blood units served as negative controls; two were filtered (one with each type of WBC-reduction filter) and one remained unfiltered. All red cell units were then stored at 4 degrees C for 42 days. One of the five filtered red cell units (20%) in each filter group supported growth of Y. enterocolitica. In contrast, 4 (100%) of 4 unfiltered inoculated red cell units had growth (p = 0.04). Overall, 2 (20%) of 10 units of WBC-reduced red cells supported the growth of Y. enterocolitica, as compared to 100 percent of unfiltered red cell units after inoculation (p = 0.015). Bacterial contamination was not detected in any of the three noninoculated units. It can be concluded that prestorage WBC filtration significantly reduces the potential for growth of Y. enterocolitica in red cells stored at 4 degrees C for 42 days.

Visual identification of bacterially contaminated red cells.

There have been increasing numbers of reports of transfusion-acquired Yersinia enterocolitica bacteremia (including several fatal cases). Fifteen units of whole blood were inoculated with various concentrations of Y. enterocolitica serotype 0:3 and processed into AS-3 preserved red cells (RBCs). Consistent growth of the organism was found at inoculum concentrations greater than or equal to 10 colony-forming units per mL. In all 13 units of RBCs that supported the growth of Y. enterocolitica, a darkening in color (due to hemolysis and a decrease in pO2) was observed in the bag. The attached sample segments, which were sealed from the main unit, remained sterile and did not darken. This color change was apparent in all the contaminated units by Day 35, which was 1.5 to 2 weeks after the bacteria were first detected in cultures of the blood. Hence, by comparison of the color of the segment tubing with that of the unit itself, units grossly contaminated with Y. enterocolitica can be identified prior to transfusion. Moreover, review of photographs on file at the Centers for Disease Control revealed this dramatic color change in 2 units of blood that caused transfusion-transmitted sepsis (Enterobacter agglomerans and an unidentified gram-negative bacillus, not Yersinia sp.), which demonstrated that the color change was not limited to Y. enterocolitica. This method of visual identification of contaminated units of blood could decrease the incidence of posttransfusion bacterial sepsis.

An animal model of allogeneic donor platelet refractoriness: the effect of the time of leukodepletion.

Approximately 50% of multi-transfused individuals become refractory to random donor platelets. Recent clinical data suggest that those patients receiving leukocyte-depleted blood products are less likely to become refractory to random donor platelets than recipients of non-leukocyte-depleted products. Leukocyte depletion can be performed immediately after collection of a unit of whole blood before its storage (prestorage leukodepletion) or just before the transfusion of the blood product to a recipient, after its storage (poststorage leukodepletion). However, the most appropriate time for the leukodepletion of blood products has not been established. The present study was undertaken to establish an animal model of allogeneic platelet refractoriness, and to compare the effect of prestorage and poststorage leukodepletion on the frequency of refractoriness to allogeneic donor platelets. In this model, two strains of rabbits were used: California Black rabbits were used as blood donors, while New Zealand White rabbits were used as recipients. Eight weekly infusions of nonleukodepleted allogeneic fresh blood resulted in an allogeneic platelet refractory rate of 91.2% (31/34). The prestorage leukodepletion of the donor blood was associated with a significantly higher allogeneic platelet survival and lower refractory rate (33.3%) to allogeneic platelets than poststorage leukodepletion (66.7%). Furthermore, the data suggest that cell-free plasma products are capable of inducing refractoriness to allogeneic donor platelets; the stored plasma having a greater likelihood of inducing such refractoriness than fresh plasma. Thus, these data provide evidence that the prestorage leukodepletion of allogeneic donor blood is associated with a lower frequency of refractoriness and better allogeneic platelet survival than poststorage leukodepletion.

Preparation of white cell-depleted red cells for 42-day storage using an integral in-line filter.

A new blood pack system for the preparation of white cell-depleted red cells was studied. The system is a modified additive-solution quadruple-unit blood pack that incorporates a cellulose-acetate fiber depth filter in-line between the AS-3 additive bag and the CP2D collection bag. Mean +/- SD white cell removal from 156 units processed under standard production conditions was 97.7 +/- 2.7 percent; residual white cells were 1.1 +/- 1.0 x 10(8) per unit. Red cell loss was 10.0 +/- 1.0 percent (n = 43). Mean platelet removal was 80.9 percent from units from which platelet concentrates were not prepared (n = 47). Microaggregates did not form during storage, and hemolysis of filtered red cells was lower than that of unfiltered controls. Filtered AS-3 red cells stored for 42 days had a 51Cr survival of 80.1 +/- 5.7 percent (mean +/- SD) as compared with 78.9 +/- 6.2 percent for unfiltered controls (n = 17). This in-line filter system provides white cell-depleted, microaggregate-free red cells that can be stored for 42 days.

Ascorbate-2-phosphate in red cell preservation. Clinical trials and active components.

A red cell additive solution (AS-005) containing ascorbate-2-phosphate (AsP) to maintain 2,3-diphosphoglycerate, plus adenine, phosphate, and mannitol to retain viability and reduce hemolysis, was evaluated by human clinical trials. A crossover design was used with another additive solution (Nutricel AS-3, Cutter Laboratories) serving as the control for each donor. Each additive solution was evaluated at 35 and 42 days of storage. There was no significant difference between the red cell viability of the two storage solutions at either time period. Split-bag, AS-005 in vitro studies at two temperatures (2.5 and 5.5 degrees C), both within the range of 1 to 6 degrees C approved by the American Association of Blood Banks and the Food and Drug Administration, resulted in dramatically different in vitro parameters, including a threefold difference in 2,3-diphosphoglycerate (2,3-DPG), a fivefold difference in glucose, and significant differences in pH and adenosine triphosphate. High-pressure liquid chromatography data confirmed the preliminary report that 1 to 2 percent (wt/wt) oxalate was present in preparations of AsP. In vitro storage data confirmed that oxalate is the active component of AsP that preserves 2,3-DPG during storage.

Five-week red cell storage with preservation of 2,3 DPG.

The 2,3 diphosphoglycerate (2,3 DPG) content of red cells stored in current anticoagulant-preservative products decreases rapidly after the first few days of storage, and by 3 weeks the red cells are essentially depleted of 2,3 DPG. Because ascorbic acid and ascorbate-2-phosphate (A-2-P) are effective in maintaining erythrocyte 2,3 DPG during liquid preservation, ascorbate was stabilized through autoclaving and subsequent storage by adding it as the trisodium salt of A-2-P to a phosphate-adenine-saline solution at a pH of 8.5 to 9.0. Red cell concentrates prepared from blood drawn into citrate-phosphate-double-dextrose were supplemented with the A-2-P additive solution (AS-4) and studied in vitro and in vivo. Mean 2,3 DPG values for 22 units were 147.6, 113.5, and 82.3 percent of initial value after storage for 3, 4, and 5 weeks, respectively. Maintenance of 2,3 DPG was at the expense of adenosine triphosphate (ATP), which fell to as low as 22.2 percent of initial value after 5 weeks. Despite the low ATP values, the 24 hour 51Cr-labeled red cell recoveries averaged 80.8 and 74.1 percent after 4 and 5 weeks of storage, respectively. The AS-4 system provides a red cell product with acceptable viability and improved oxygen off-loading function.

Storage of platelets on flatbed agitators in polyvinyl chloride blood bags plasticized with tri(2-ethylhexyl) trimellitate.

Data are presented showing that platelets in polyvinyl chloride blood bags plasticized with tri(2-ethylhexyl) trimellitate can be stored on flatbed agitators (shakers) without the pH falling below 6.0. The lowest pH seen after 7 days of storage in 46 units with platelet yields ranging from 3.6 to 13.3 X 10(10) per unit was 6.43. These bags have a O2 transmission rate of 13.3 mumol per hour per bag. Platelet bags with a O2 transmission rate of 7.9 mumol per hour per bag experience a pH fall after 5 days of storage on the shaker in units whose platelet yield on average exceeds 10 X 10(10). Platelets can be stored on first-generation shakers (70 cycles/min, stroke = 1 inch) without an attempt at manual resuspension of the platelet button. The count after 30 minutes on the shaker averaged 89 +/- 15 percent of the expected count, indicating that resuspension was nearly complete after a relatively short period. Red cells, but not platelets, settled out during storage on the shaker.

pH changes caused by bacterial growth in contaminated platelet concentrates.

While platelet concentrates are stored at room temperature, lactic and other acids are produced and the pH decreases as the buffering capacity of the plasma is exhausted. Platelet viability will be compromised if the pH decreases to pH 6.0 and below. Similarly, a pH decrease can be produced also by bacterial contamination if the organisms produce acid as an end product. Thus the determination of pH could serve as a sensitive indicator of bacterial contamination. This hypothesis was tested by us by inoculating known organisms into platelet concentrations. It was found that the pH may decrease, may remain unchanged, or, in a few cases, even increase. Visual signs of contamination could be observed but not consistently enough to be entirely dependable. Therefore, this method does not appear to detect bacterial contamination reliably in platelet concentrates.

Factors affecting white cell content in platelet concentrates.

In this study, we investigated the factors affecting white cell content in platelet concentrates. White cell yields can be reduced 50 percent by stopping platelet-rich plasma expression when the interface is 1 cm from the top of the blood bag as compared to stopping expression when the interface reaches the top of the bag. Further reductions can be achieved by careful handling during transfer of units from the centrifuge cups to expressors (after the first spin) and by carefully balancing units against each other to ensure proper rotor balance during the first spin. Following these suggestions, blood banks should be able to produce platelet concentrates with white cell yields between 2 and 6 X 10(7) and with platelet yields between 7.5 and 8 X 10(10). Transfusion of this product may reduce febrile reactions and lower the incidence of alloimmunizations.

In vitro function and in vivo viability of stored platelet concentrates. Effect of a secondary plasticizer component of PVC storage bags.

Previous studies have shown that CL-2399 (Cutter) and PL-130 (Fenwal) polyvinyl chloride (PVC) plastic bags are unsatisfactory for storage of platelet concentrates (PC) at 22 degrees C. In an effort to explain the effects of plastic bags, the chemical make-up of CL-2399 and PL-130 PVC films was determined and compared with that of P1-146 (Fenwal) PVC, which is satisfactory for PC storage at 22 degrees C. The only significant difference between the three materials was the incorporation of tetrahydrofurfuryl oleate (THFO) as a secondary plasticizer in CL-2399 and PL-130. The response of platelets to aggregating agents, uptake of serotonin, recovery from hyptonic stress, and serotonin release during storage following storage in a modified CL-2399 plastic prepared without THFO and designated CL-3000 (Cutter) was equivalent to PL-146 and far superior to CL-2399. In vivo studies in two laboratories of platelets stored in CL-3000 bags showed satisfactory recovery (56 +/- 4.2% and 46.7 +/- 2.7%) and survival (6.4 +/- 0.4 days and 7.4 +/- 0.6 days). From these studies we conclude that the THFO secondary plasticizer component of PL-130 and CL-2399 is the cause of the poor platelet viability of platelets stored in these plastic bags. The mechanism of impairment is not known. The causative agent(s) may be degradation products of THFO (formed during manufacture of the PVC film) that are leached from the plastic into PC during storage.