The effects of polyvinyl chloride and polyolefin blood bags on red blood cells stored in a new additive solution.

BACKGROUND AND OBJECTIVES: Red blood cells (RBCs) must be stored in polyvinyl chloride (PVC) bags plasticized with di-2-ethylhexyl phthalate or a similar plasticizer to achieve their full storage life with conventional storage solutions. Improved storage solutions might remove this requirement and allow blood storage in other plastics. Experimental Additive Solution-61 (EAS-61), which maintains RBCs for 9 weeks with reduced haemolysis and satisfactory 51Cr 24-h recovery, is an appropriate candidate improved RBC storage solution. MATERIALS AND METHODS: Twenty-four units of packed RBCs were pooled in groups of four units, each pool was realiquoted into four units and stored, six pooled units per arm, in one of the following: 100 ml of EAS-61 in PVC; 200 ml of EAS-61 in PVC; 100 ml of EAS-61 in polyolefin (PO); and 200 ml of EAS-61 in PO. Haemolysis, RBC morphology indices, RBC ATP concentrations, and other measures of RBC metabolism and function were measured weekly. RESULTS: RBC haemolysis exceeded 1% by 7 weeks in PO bags containing 100 ml or 200 ml of EAS-61. In PVC bags, haemolysis was less than 1% at 11 weeks. RBC ATP concentrations were 1 mol/g of haemoglobin (Hb) higher at 2 weeks in the PVC-stored units. CONCLUSIONS: RBCs stored in PVC had markedly less haemolysis and higher RBC ATP concentrations than those stored in PO. Haemolysis would limit RBC storage in PO bags to a duration of 6 weeks, even with EAS-61.

The role of electrolytes and pH in RBC ASs.

BACKGROUND: Experimental additive solutions (EASs) containing saline, adenine, glucose, mannitol and disodium phosphate can support RBCs for 9 or 10 weeks if used in 200- or 300-mL volumes. The effects of variations in the electrolyte composition and volume of EASs were explored. STUDY DESIGN AND METHODS: In three four-arm studies, 24 RBC units were pooled in groups of 4 and realiquoted as test units to ensure that all donors were equally represented in each study arm. In Study 1, units were stored for 11 weeks in EAS containing 0, 10, 20, or 30 mmol per L of sodium bicarbonate. In Study 2, units were stored for 9 weeks in EAS containing 26, 50, 100, or 150 mmol per L of sodium chloride. In Study 3, units were stored in 100 or 200 mL of AS-3 or EAS-61. RBC ATP concentrations and hemolysis were measured weekly. RESULTS: Increasing the sodium bicarbonate content of EASs increased the pH throughout storage and increased RBC ATP concentrations in the later phases of storage, but it had no effect on hemolysis. Increased sodium chloride content of EASs led to lower RBC ATP concentrations and increased hemolysis. In EAS-61, RBC ATP concentrations were increased throughout storage, and hemolysis was lower than that of RBCs stored in AS-3. CONCLUSION: RBC ATP synthesis is highly dependent on the pH of the AS. Hemolysis is affected by the salt content and volume of the AS.

The effect of two additive solutions on the postthaw storage of RBCs.

BACKGROUND: Sterile systems for freezing and for washing thawed blood will allow the storage of RBCs for more than 24 hours after removal of the cryoprotectant glycerol. This study assessed the effect of two ASs in maintaining deglycerolized RBCs. STUDY DESIGN AND METHODS: Twenty-four RBC units were stored for 6 days, pooled in groups of 4, realiquoted, sterilely glycerolized, and frozen. One month later, the units were thawed, sterilely deglycerolized by using an automated system (H215; Haemonetics), and stored for 5 weeks in either 100 or 200 mL of AS-3 or an experimental AS (EAS-61). Sterile samples were taken weekly for chemical and morphometric analysis. RESULTS: The glycerolization and deglycerolization process produced highly comparable RBC units, but it caused a marked reduction of RBC pH, to about 6.4 at the beginning of storage. The addition of acidic AS-3 further reduced the pH, which in turn reduced glucose consumption, lactate formation, and RBC ATP concentrations. Alkaline EAS-61 increased these measures. Hypotonic EAS-61 caused increased cell swelling and hemolysis, despite better RBC morphology. CONCLUSIONS: Automation of sterile glycerolization and deglycerolization with the H215 works well, but the solutions should be reformulated for extended postthaw storage. This would best be accomplished by raising the pH of the wash solutions by the addition of disodium phosphate or sodium bicarbonate or both, by using alkaline ASs, and by matching the osmolality of the wash solution and ASs.

A multicenter study of in vitro and in vivo values in human RBCs frozen with 40-percent (wt/vol) glycerol and stored after deglycerolization for 15 days at 4 degrees C in AS-3: assessment of RBC processing in the ACP 215.

BACKGROUND: The FDA has approved the storage of frozen RBCs at -80 degrees C for 10 years. After deglycerolization, the RBCs can be stored at 4 degrees C for no more than 24 hours, because open systems are currently being used. Five laboratories have been evaluating an automated, functionally closed system (ACP 215, Haemonetics) for both the glycerolization and deglycerolization processes. STUDY DESIGN AND METHODS: Studies were performed at three military sites and two civilian sites. Each site performed in vitro testing of 20 units of RBCs. In addition, one military site and two civilian sites conducted autologous transfusion studies on ten units of previously frozen, deglycerolized RBCs that had been stored at 4 degrees C in AS-3 for 15 days. At one of the civilian sites, 10 volunteers received autologous transfusions on two occasions in a randomized manner, once with previously frozen RBCs that had been stored at 4 degrees C in AS-3 for 15 days after deglycerolization and once with liquid-preserved RBCs that had been stored at 4 degrees C in AS-1 for 42 days. RESULTS: The mean +/- SD in vitro freeze-thaw-wash recovery value was 87 +/- 5 percent; the mean +/- SD supernatant osmolality on the day of deglycerolization was 297 +/- 5 mOsm per kg of H(2)O, and the mean +/- SD percentage of hemolysis after storage at 4 degrees C in AS-3 for 15 days was 0.60 +/- 0.2 percent. The paired data from the study of 10 persons at the civilian site showed a mean +/- SD 24-hour posttransfusion survival of 76 +/- 6 percent for RBCs that had been stored at 4 degrees C for 15 days after deglycerolization and 72 +/- 5 percent for RBCs stored at 4 degrees C in AS-1 for 42 days. At the three sites at which 24-hour posttransfusion survival values were measured by three double-label procedures, a mean +/- SD 24-hour posttransfusion survival of 77 +/- 9 percent was observed for 36 autologous transfusions to 12 females and 24 males of previously frozen RBCs that had been stored at 4 degrees C in AS-3 for 15 days after deglycerolization. CONCLUSION: The multicenter study showed the acceptable quality of RBCs that were glycerolized and deglycerolized in the automated ACP 215 instrument and stored in AS-3 at 4 degrees C for 15 days.

RBC storage for 11 weeks.

BACKGROUND: Increasing the length of RBC storage can increase both RBC availability and quality. This work addresses 11-week RBC storage in experimental ASs (EASs). STUDY DESIGN AND METHODS: Three studies were performed. In the first, 24-hour in vivo recovery of (51)Cr-labeled autologous RBCs was measured in nine volunteers after storage of their RBCs for 11 weeks in EAS 67. In the second study, 4 units of blood were divided and stored in aliquots with an EAS containing 0, 15, 30, or 45 mmol per L of mannitol; then hemolysis, RBC morphology, and microvesicle protein were measured. In the third study, 6 full units were stored for 12 weeks in the EAS containing 30 mmol per L of mannitol, with weekly sampling for morphologic and biochemical measures of RBC quality. RESULTS: RBCs stored for 11 weeks in EAS-67 had a mean 24-hour in vivo recovery of 79 +/- 5 percent, but the hemolysis was 1.35 +/- 0.68 percent. Increasing mannitol content of the EAS reduced hemolysis but increased microvesiculation. EAS-76, with 30 mmol per L of mannitol allowed 11-week storage with 0.48 +/- 0.10 percent hemolysis at 11 weeks and 0.62 +/- 0.14 percent hemolysis at 12 weeks. CONCLUSION: It is possible to store RBCs for 11 weeks in EAS with greater than 75 percent recovery and less than 1 percent hemolysis.

A hypotonic storage solution did not prolong the viability of RBCs.

BACKGROUND: Hypotonic storage solutions and WBC filtration are both reported to improve RBC viability. This study tested the ability of an investigational hypotonic storage solution (AS-24, Medsep Corp.) to extend the viability of liquid-stored RBCs to 8 weeks. STUDY DESIGN AND METHODS: In a pair of crossover trials, 11 RBC units, WBC-reduced by filtration and stored in AS-24 for 8 weeks, were compared with units from the same donors that were stored for 6 weeks in AS-3, and 13 RBC units, WBC-reduced by filtration and stored in AS-3 for 8 weeks, were compared with units from the same donors that were stored for 6 weeks in AS-3. Viability was measured by the (51)Cr/(99m)Tc double-isotope method. RESULTS: RBC viability at 8 weeks averaged 64 +/- 3 percent in the AS-24 units and 67 +/- 2 percent in the AS-3 units. It was equal at 77 +/- 3 percent and 77 +/- 2 percent after 6 weeks' storage in AS-3 in both trials. CONCLUSIONS: Prestorage WBC reduction and storage in AS-24 did not extend RBC viability to 8 weeks. The improved viability previously demonstrated with storage of dilute suspensions of RBCs in hypotonic solutions is probably caused by factors other than the hypotonicity.

The effects of phosphate, pH, and AS volume on RBCs stored in saline-adenine-glucose-mannitol solutions.

BACKGROUND: RBC ATP concentrations are the most important correlate of RBC viability. Tests were performed to determine whether increased AS volume, pH, and phosphate content increased stored RBC ATP concentrations. STUDY DESIGN AND METHODS: In three studies, packed RBCs were pooled in groups of 3 or 4 units and realiquoted as combined units to reduce intradonor differences. Pooled units were stored in the licensed ASs, AS-1 or AS-5, which contain saline, adenine, glucose, and mannitol (SAGM), or in experimental ASs (EASs) containing SAGM and disodium phosphate. Ten pools were stored in AS-1 at RBC concentrations equivalent to 100, 200, or 300 mL of AS. Six pools were stored in 100, 200, 300, or 400 mL volumes of EAS-61. Ten pools were stored in 100 mL of AS-5, 200 mL of EAS-61, or 300 mL of EAS-64. RBC ATP concentration and other measures of RBC metabolism and function were measured weekly. RESULTS: RBC ATP concentrations decreased sooner with storage in increasing volumes of AS-1. In EAS-61 and EAS-64, RBC ATP concentrations initially increased and stayed elevated longer with increasing AS volume. CONCLUSIONS: The addition of disodium phosphate to SAGM AS increases the RBC ATP concentrations. Reducing storage Hct appears to have a separate beneficial effect in reducing hemolysis.

Psoralen photochemical inactivation of Orientia tsutsugamushi in platelet concentrates.

BACKGROUND: The risk of transfusion transmission of disease has been reduced by the combination of predonation questions and improved transfusion-transmitted disease assays, but the risk is still present. This study was conducted to determine if psoralen photochemistry could inactivate an obligate intracellular bacterium, with documented potential for transfusion, in PCs to further improve safety. STUDY DESIGN AND METHODS: PCs were inoculated with MNCs infected with Orientia tsutsugamushi. The concentrates were treated with amounts ranging from 0.86 to 138 micromol per L of 4'-(aminomethyl)-4,5',8-trimethylpsoralen hydrochloride (AMT) combined with a constant long-wave UVA light (320-400 nm) exposure of 5 J per cm(2). The effects of photochemical treatment were analyzed by using a mouse infectivity assay along with in vitro testing by PCR, indirect fluorescence antibody, direct fluorescence antibody, and Giemsa staining. RESULTS: AMT, at 0.86 micromol per L or more, combined with UVA light of 5 J per cm(2), inactivated O. tsutsugamushi that contaminated PCs. The PCs that did not receive the combined treatment caused infection. CONCLUSIONS: The psoralen AMT, in conjunction with UVA light exposure, effectively abolished the infectivity of PCs deliberately contaminated with the scrub typhus organism O. tsutsugamushi, as tested in a mouse infectivity assay.

The viability of autologous human red cells stored in additive solution 5 and exposed to 25 degrees C for 24 hours.

BACKGROUND: No data exist on the viability of red cells (RBCs) stored in modern additive solution systems and allowed to warm above 10 degrees C. STUDY DESIGN AND METHODS: In a randomized crossover study, 3 units of blood were collected at least 8 weeks apart from 11 volunteer donors and stored in additive solution 5 (AS-5). Of 3 units from each volunteer, 1 was stored for 6 weeks at 4 degrees C, 1 for 5 weeks at 4 degrees C except for 24 hours at 25 degrees C on Day 14, and 1 for 5 weeks at 4 degrees C except for 24 hours at 25 degrees C on Day 28. Units were sampled periodically during storage; at the end of storage, viability was measured by the 99mTc/51 Cr double-label method. RESULTS: RBC viability was not significantly different in the storage protocols. Less than 1 percent of stored cells hemolyzed. RBC ATP concentrations at the end of storage correlated with viability and were approximately equal in the warmed units after 30 days' storage and the conventionally stored units after 42 days. CONCLUSIONS: The data suggest that RBCs stored in AS-5 and allowed to warm to 25 degrees C for 24 hours lose about 12 days of their shelf life.

Effect of 24-hour storage at 25 degrees C on the in vitro storage characteristics of CPDA-1 packed red cells.

BACKGROUND: Packed red cells (RBCs) warmed above 10 degrees C are generally discarded. Few data exist on the degree of accelerated metabolism and increased hemolysis of packed RBCs allowed to warm. STUDY DESIGN AND METHODS: Twenty-four CPDA-1 packed RBC units were combined in 3-unit pools and subdivided into 2 test units and a control unit. One test unit from each pool was warmed to 25 degrees C for 24 hours on Day 6 and the other test unit was warmed on Day 20; control units were maintained at 1 to 6 degrees C. RBC and supernatant chemistries and RBC morphology were measured weekly (Days 0, 7, 14, 21, and 28) and on the day before warming (Days 6 and 20). RESULTS: Warming CPDA-1 packed RBCs accelerated the catabolism of glucose 10-fold and produced concentrations of glucose, lactate, and ATP after 25 days of storage that were equivalent to those in unwarmed units at 35 days. Supernatant sodium and potassium concentrations were corrected partially with warming. RBC morphology transiently normalized with warming and without increased hemolysis; no bacteria growth was detected. CONCLUSION: One day of 25 degrees C storage of CPDA-1 packed RBCs accelerates essential metabolite break-down equivalent to 10 days of storage at 1 to 6 degrees C. It does not appear to matter whether the packed RBCs are warmed on Day 6 or Day 20. This information may be useful in determining the acceptability of blood allowed to warm above 10 degrees C.

An improved process for the production of sterile modified haemoglobin solutions.

The process for manufacturing bulk quantities of sterile solutions of human haemoglobin (Hb) cross-linked between the alpha chains (alphaalphaHb) with bis(3,5-dibomosalicyl) fumarate (DBBF) was modified to: (1) improve product purity; (2) increase product yield; (3) eliminate non-United States Pharmacopoeia materials; (4) reduce reagent costs; and (5) reduce production time. These process modifications were the result of increased scientific understanding of the Hb cross-linking chemistry and were in the form of engineering and procedure controls that reflect current good manufacturing practices (cGMP). Purity, as reflected in the fractional yield of the desired alphaalphaHb product, has increased from 60% to 90+% of total Hb, and uncross-linked Hb was virtually eliminated. Impurities such as pyrogens, methaemoglobin, phospholipid, and free iron were reduced. The net yield of alphaalphaHb was increased from 33% to 58% of starting Hb content. Production time, the use of overtime, the consumption of expensive reagents and filters, and losses because of contamination have all been reduced. As a result, cost per gram of alphaalphaHb produced has decreased 60%. With this improved process, efficient production of very pure alphaalphaHb is possible.

In vitro and in vivo persistence of reticulocytes from donor red cells.

BACKGROUND: Reticulocytes are important in the phenotyping of transfused patients. Reticulocytes can persist in blood units for the shelf life of the unit. STUDY DESIGN AND METHODS: Temperature dependence of reticulocyte persistence was examined in vitro at 4, 24, and 37 degrees C by using thiazole orange staining and flow cytometric analysis. Two-color flow cytometric analysis was used to evaluate the persistence of donor reticulocytes in transfused patients. RESULTS: Flow cytometric analysis using thiazole orange demonstrated that persistence of reticulocytes in units of stored CPDA-1 blood was temperature-dependent. Reticulocytes disappeared over 13 and 6 days at 24 degrees C and 37 degrees C, respectively, but at 4 degrees C the reticulocyte count changed little over 35 days. Two-color flow cytometric analysis of reticulocyte antigens was used to follow donor reticulocytes in 14 transfusion events in nine different patients. Donor reticulocytes persisted through 24 hours in 75 percent of the patients and were detectable at 48 hours in three patients. CONCLUSION: This study demonstrates that reticulocytes persist during refrigerated storage; they are detectable in the circulation of most recipients for the first 24 hours after transfusion and in the circulation of a few recipients after 48 hours. These findings may have relevance for separation techniques based on reticulocyte density in samples drawn shortly after transfusion and for evaluation of reticulocyte counts in patients with hematologic abnormalities.

A flow cytometric method for phenotyping recipient red cells following transfusion.

BACKGROUND: Reticulocyte phenotyping is used for transfused patients, who have red cell antibodies, to match blood for subsequent transfusion. Current methods are labor-intensive and require a significant amount of sample. STUDY DESIGN AND METHODS: A simple dual-color flow cytometry method developed for antigen typing of reticulocytes in mixed red cell populations is reported. Antigens were labeled by an indirect immunofluorescence technique using undiluted reagent sera as the primary label, biotinylated goat anti-human IgG as the secondary label, and avidin-phycoerythrin as the fluorescent stain. Reticulocytes were labeled with a thiazole orange fluorescent stain. Reticulocyte identification and antigen typing were performed on 319 samples to establish the validity of the procedure. Mixed red cells were prepared in all possible c antigen combinations to simulate transfusion concentrations of 25, 50, and 75 percent. RESULTS: The anti-c flow cytometry profiles readily distinguished between antigen-positive and antigen-negative populations and allowed the detection of reticulocytes at all simulated transfusion concentrations. Similar results were obtained in experiments using C, K, s, Fya, Fyb, Jka, or Jkb sera against equal volumes of antigen-positive and -negative cells. Anti-S gave inconsistent results. The in vitro results were confirmed in 19 transfused patients who had received red cells antigenically different from their own as well as cells from 1 chimera blood donor. CONCLUSION: This method provides a simpler, safer, less labor-intensive, and less subjective technique requiring far less sample volume than current methods for antigen typing of reticulocytes in mixed red cell samples from recently transfused patients.

Relationship of major histocompatibility complex class II genes to inhibitor antibody formation in hemophilia A.

Approximately 14% of transfused hemophiliacs develop an anti-factor VIII inhibitory antibody which specifically neutralizes factor VIII procoagulant activity. In this study an association of the major histocompatibility complex (MHC) with inhibitor antibody formation was evaluated by restriction fragment length polymorphism (RFLP) analysis using BamHI, EcoRI, HindIII, PstI, PvuII and TaqI digested genomic DNA probed with DP beta, DQ alpha, DQ beta and DR beta class II MHC gene probes. The RFLP patterns for 16 non-inhibitor and 11 inhibitor hemophiliac patients were analyzed. These 24 enzyme:probe combinations generated 231 fragments. Fifteen (15) fragments associated with the inhibitor phenotype; odds ratios ranged from 5.1 to 45 and lower bounds of 95% confidence intervals were greater than 1.000 for all 15 fragments. Five (5) fragments associated with non-inhibitors, with odds ratios ranging from 6.4 to 51.7. This report establishes a MHC related genetic basis for the inhibitor phenotype. No statistically significant differences in the distribution of serologically defined HLA-DR phenotypes were observed between the inhibitor and non-inhibitor groups.