Duck hepatitis B photoinactivation bydimethylmethylene blue in RBC suspensions

BACKGROUND: Dimethylmethylene blue (DMMB) has been used to photoinactivate a number of model viruses, including VSV, in RBC suspensions under conditions that preserve in vitro RBC properties during storage. The relative sensitivity of duck HBV (DHBV) and VSV to photoinactivation by DMMB was investigated by performing an indirect immunofluorescence assay (IFA) using primary duck hepatocyte (PDH) cultures or a standard plaque assay for the respective viruses. STUDY DESIGN AND METHODS: DMMB was added to 45-percent Hct, WBC-reduced, oxygenated AS-3 RBCs at 10-, 1-, and 0.1-microM concentrations. Samples (1-mm thick) were illuminated with 5.4-mW per cm(2) of red light for 2 or 9 seconds. Unilluminated samples without DMMB or with 10 microM DMMB served as control. RESULTS: DHBV and VSV were rapidly photoinactivated by DMMB in a concentration and light-dose-dependent fashion. Neither virus was substantially inactivated by incubation with DMMB in the dark. For a given light exposure, DHBV required a concentration of DMMB one-one hundredth that of VSV to achieve approximately the same level of inactivation. CONCLUSION: DHBV appears to be considerably more sensitive than VSV to DMMB photoinactivation. Photoinactivation in 45-percent Hct RBCs can be achieved in seconds by using micromolar quantities of dye.     

Thiazole orange, a DNA-binding photosensitizer with flexible structure, can inactivate pathogens in red blood cell suspensions while maintaining red cell storage properties

BACKGROUND: Development of a robust pathogen reduction system for red cells (RBCs) utilizing photosensitive dyes has been constrained by hemolysis, usually mediated by reactive oxygen species emanating from dye free in solution as well as dye bound to the RBC membrane. The RBC binding properties of thiazole orange (TO), a flexible nucleic acid intercalating cyanine dye that predominantly acts as a photosensitizer only when bound, were assessed along with its virucidal, bactericidal, and light-induced hemolytic activities. STUDY DESIGN AND METHODS: Leukodepleted 20% hematocrit RBCs suspended in Erythrosol (RAS-2) were oxygenated, inoculated with test organisms, incubated with TO, and illuminated. Control and treated samples were analyzed by appropriate assay. Identically prepared, but uncontaminated samples were phototreated, concentrated to 45% hematocrit, and assayed for potassium leakage, hemolysis, and ATP during storage. RESULTS: Approximately 21 percent TO bound to RBCs. Phototreatment inactivated from 5.4 to 7.1 log(10) of 5 tested viruses and from 2.3 to greater than 7.0 log(10) of 8 tested bacteria. Phototreated RBCs exhibited only slightly increased hemolysis, moderately elevated potassium efflux, and similar levels of ATP compared to controls. CONCLUSION: TO can photoinactivate several model viruses and pathogens in RBCs under conditions that produce limited hemolysis without the addition of quenchers or competitive inhibitors.     

The band III ligand dipyridamole protects human RBCs during photodynamic treatment while extracellular virus inactivation is not affected

BACKGROUND: Recently, the potential usefulness of dipyridamole (DIP) in protecting RBCs against the harmful side effects of photodynamic sterilization was demonstrated. In the present study, the use of DIP for selective protection of RBCs was investigated under conditions more relevant for blood bank practice. STUDY DESIGN AND METHODS: WBC-reduced RBC suspensions (30% Hct) were treated with 1,9-dimethylmethylene blue and red light, and the influence of the inclusion of DIP on photohemolysis was assessed as a function of sensitizer concentration, light dose, and storage time. Furthermore, the possible interference of DIP with inactivation of extracellular virus by use of a panel of different viruses (HIV-1, pseudorabies virus [PRV], bovine viral diarrhea virus [BVDV], VSV, encephalomyocarditis, and canine parvovirus) was investigated. RESULTS: In WBC-reduced RBC suspensions (30% Hct), DIP exerted a clear protective effect against photohemolysis. Part of this protection was achieved with concentrations near the dissociation constant for band III binding. Importantly, efficiency of inactivation of extracellular HIV-1, PRV, BVDV, and VSV was not significantly impaired by the inclusion of DIP. Phototreatment conditions, resulting in a 4 to 5 log inactivation of extracellular HIV-1 and PRV, resulted in a high level of hemolysis after 28 days of storage. This long-term hemolysis could be decreased, but not completely prevented, by the inclusion of DIP. CONCLUSION: Photohemolysis in RBC concentrates can be reduced substantially by the application of DIP, while the efficacy of inactivation of HIV-1 and other viruses remains unchanged.     

Positively charged porphyrins: a new series of photosensitizers for sterilization of RBCs

BACKGROUND: Photodynamic treatment could be a way to inactivate pathogens in RBCs. The objective of this study was to characterize the virucidal activity and RBC-damaging activity of a series of cationic porphyrins. Using the most efficacious photosensitizer, various in-vitro human RBC quality variables and in-vivo RBC survival in Rhesus monkeys were evaluated. STUDY DESIGN AND METHODS: RBCs, spiked with 5 log of extracellular VSV, were treated with porphyrins (25 micro mol/L) and red light (100 W/m2) and essayed for virucidal activity. In-vitro RBC quality variables were assessed during 5 weeks of storage in various ASs. In-vivo survival was investigated with autologous RBCs in Rhesus monkeys. RESULTS: Tri-P(4) was by far the best sensitizer of a series tested, giving the least hemolysis under conditions that resulted in 5 log-kill of extracellular VSV. Under our experimental conditions, the percentage hemolysis in treated cells was 5.1 +/- 1.1 percent after 5 weeks of storage in SAG-M compared to 1.9 +/- 1.1 percent in the untreated control. Storage in AS-3 resulted hemolysis of 2.3 +/- 1.9 percent. With the exception of IgG binding and potassium leakage, RBC quality variables remained unchanged after photodynamic treatment. Addition of reduced glutathione (GSH) during treatment reduced IgG binding. The 24-hour recovery and T50 of treated RBCs in Rhesus monkeys were satisfactory. CONCLUSION: Porphyrin Tri-P(4) may be a suitable photosensitizer for sterilization of RBCs. However, further exploration to optimize the method is necessary to reach clinically acceptable goals.     

Preservation of red cell properties after virucidal phototreatment with dimethylmethylene blue

BACKGROUND: All published reports have described methods for virus photoinactivation which significantly alter red cell (RBC) properties during storage. In order to improve virucidal activity and reduce damage to RBCs, a series of phenothiazine derivatives were either synthesized or purified and screened for bacteriophage inactivation and red cell potassium efflux. One compound, 1,9-dimethylmethylene blue (dimethyl-methylene blue), had superior screening results and was chosen for further characterization. STUDY DESIGN AND METHODS: White cell reduced RBC suspensions (30% hematocrit) were deliberately inoculated with extracellular virus or virus-infected VERO cells, incubated with 4 microM dimethyl-methylene blue and illuminated with cool-white fluorescent light. Control and treated samples were titered for virus inactivation. In parallel studies, RBC suspensions were exposed to dimethylmethylene blue and light under identical conditions and assayed for in vitro RBC storage properties. RESULTS: Phototreatment of RBC suspensions inactivated > 4.4 log10 of extracellular vesicular stomatitis virus (VSV), > 3.0 log10 of intracellular VSV, > 5.0 log10 of extracellular pseudorabies virus (PRV), > 4.8 log10 of intracellular PRV, > 4.7 log10 of extra-cellular bovine virus diarrhea virus, 5.8 log10 of bacterio-phage phi 6 and > 7 log10 of bacteriophage R17. Encephalo-myocarditis virus, a nonenveloped picornavirus, was resistant to photoinactivation. Virucidal conditions resulted in no detectable IgG binding in 11 of 13 samples, unchanged RBC morphology, normal banding patterns of RBC membrane proteins on SDS PAGE, and unaltered characteristics of 12 of 13 RBC antigens during storage as measured by antibody titrations. In addition, minimal changes were observed in RBC osmotic fragility, lysis, potassium efflux, ATP and 2,3-DPG levels, and the strength of one RBC antigen during storage of phototreated samples compared with controls. CONCLUSION: Dimethylmethylene blue photo-treatment can inactivate several intracellular and extracellular model viruses under conditions which minimally alter RBC properties during 42 days storage at 1–6 degrees C.     

Use of a flexible thiopyrylium photosensitizer and competitive inhibitor for pathogen reduction of viruses and bacteria with retention of red cell storage properties

BACKGROUND: Progress in developing photochemical methods for pathogen reduction of red blood cells (RBCs) has been hampered by hemolysis. A flexible, nucleic acid-intercalating thiopyrylium (TP) dye that is only photochemically active in the bound state and a competitive inhibitor of RBC membrane binding, dipyridamole (DP), was used to reduce photoinduced hemolysis stemming from free- and membrane-bound dye. STUDY DESIGN AND METHODS: Oxygenated leukodepleted 20% hct RBC suspensions were deliberately inoculated with virus or bacteria, incubated with 200 micromol per L DP and less than or equal to 100 micromol per L TP, illuminated with 1.1 J/cm(2) of red light, and titered. RBC suspensions containing 200 micromol per L DP and 160 micromol per L TP were identically phototreated, concentrated to 45% hct, and assayed for RBC storage properties. RESULTS: In RBC suspensions containing DP, TP photoinactivated vesicular stomatitis virus, pseudorabies virus, duck hepatitis B virus, bovine virus diarrhea virus, extracellular human immunodeficiency virus (HIV) to the limit of detection and 6.2 log intracellular HIV. More than 5 log inactivation of 6 bacterial species was demonstrated. DP prevented approximately 30% of TP binding to RBCs. Phototreated RBCs that were subsequently stored for 42 days exhibited acceptable levels of hemolysis, morphology scores, extracellular pH, ATP, glucose utilization rates, and lactate production. Treated samples exhibited substantially increased potassium efflux compared to controls. CONCLUSION: Use of TP photosensitizer and DP enables significant levels of pathogen reduction while retaining most, but not all RBC properties during 42 day storage.     

Laser-assisted optical rotational cell analyzer measurements reveal early changes in human RBC deformability induced by photodynamic treatment

BACKGROUND: The ability to deform is important for circulating RBCs in vivo, and earlier studies showed that this property can objectively be measured in vitro by the LORCA. In this study it was investigated whether photodynamic treatment of human RBCs (meant to inactivate contaminating pathogens) affects deformability. STUDY DESIGN AND METHODS: WBC-reduced RBC suspensions (30% Hct) were treated with 1,9-dimethylmethylene blue (DMMB) and red light. Changes in deformability were analyzed by LORCA measurements, in which elongation of the cells is measured at increasing shear stress. The effect of DMMB concentration and light dose was determined as well as the interfering effect of two scavengers of reactive oxygen species, that is, dipyridamole and Trolox. RESULTS: Photodynamic treatment with DMMB resulted in clear changes in RBC deformability. Deformability changes occurred before onset of hemolysis. Under relatively mild treatment conditions, especially deformability at low shear stress was decreased, whereas deformability changes at high shear stress only occurred under harsher treatment conditions. Inclusion of dipyridamole and/or Trolox primarily prevented deformability changes at high shear stress. CONCLUSION: LORCA measurements can effectively be used to detect changes in deformability that are induced by photodynamic treatment of human RBCs. A change in deformability represents an early marker of RBC damage under these conditions.     

Selective protection of RBCs against photodynamic damage by the band 3 ligand dipyridamole

BACKGROUND: All studied photosensitizers for virus inactivation impair RBCs. To reduce damage to the RBCs without affecting virucidal activity, selective protection of the RBCs is necessary. The ability of the band 3 ligand, dipyridamole, to react with singlet oxygen and to increase the selectivity of photosterilization was investigated. STUDY DESIGN AND METHODS: Solutions of dipyridamole were illuminated in the presence of tetrasulfonated aluminum phthalocyanine (AlPcS(4)) and dimethylmethylene blue (DMMB). Solutions of amino acids, RBCs, and vesicular stomatitis virus (VSV) in RBC suspensions were photodynamically treated in the presence or absence of dipyridamole. RESULTS: Illumination of a solution of dipyridamole in the presence of AlPcS(4) or DMMB resulted in changes in the optical spectrum of dipyridamole. The photooxidation of dipyridamole was inhibited by azide and augmented by D(2)O, which suggests the involvement of singlet oxygen. Photooxidation of amino acids and photodamage to RBCs was strongly reduced in the presence of dipyridamole. In contrast, photoinactivation of VSV in RBC suspensions was only slightly affected by dipyridamole. CONCLUSION: Dipyridamole can improve the specificity of photodynamic sterilization of RBC concentrates, thereby increasing the practical applicability of this photodecontamination method.     

A new apheresis procedure for the preparation of high-quality red cells and plasma

BACKGROUND: Multicomponent apheresis is an alternative way of preparing blood components that avoids the delay between collection and separation seen with standard whole-blood techniques. STUDY DESIGN AND METHODS: An apheresis device has been modified to facilitate the combined collection of a unit (250 mL) of red cells (RBCs) and a high-volume unit (475 mL) of plasma. The procedure, using 8-percent ACD-A, has been tested in two European blood centers. Each center performed 20 procedures for in vitro evaluation of collected RBCs and plasma and 10 procedures for evaluation of in vivo RBC recovery. All RBCs were white cell reduced by filtration. One-half of the RBC units were stored in the additive solution Adsol and one-half in another such solution (Erythro-Sol). RESULTS: The target volumes of RBCs and plasma were obtained in 27 minutes (range, 20-44 min) by using three to six cycles in a single-needle procedure. Saline (275 mL) was used to replace fluid volume withdrawn in excess of standard whole-blood donation. No side effects occurred, with the exception of minor signs of hypocalcemia. RBC ATP was well maintained (>65% at Day 42) during storage; 2,3-DPG was less well maintained, with virtually none remaining at Day 21 in either Adsol or Erythro-Sol. The RBC in vivo recoveries, after 42 days of storage at 4+/-2 degrees C determined by the single-label method, were 86.7+/-7.2 percent (Erythro-Sol) and 84.4+/-8.1 percent (Adsol). Mean plasma factor VIII levels were >100 percent in all test groups. CONCLUSION: A novel automated technique for the simultaneous collection and preparation of RBCs and plasma has been evaluated. The apheresis procedure was acceptable and well tolerated by donors, and it resulted in high-quality blood components. Further optimization of the system should yield a practicable component suitable for routine use in blood banks.     

Twelve-week RBC storage

BACKGROUND: Better storage can improve RBC availability and safety. Optimizing RBC ATP production and minimizing hemolysis has allowed progressively longer storage. STUDY DESIGN AND METHODS: In the first study, 24 units of packed CPD RBCs were pooled in groups of four, realiquoted, and added to 300 mL of one of four variants of experimental additive solution 76 (EAS-76) containing 45, 40, 35, or 30 mEq per L NaCl. Units were sampled weekly for 12 weeks for morphologic and biochemical measures. In the second study, 10 volunteers donated 2 units of RBCs for a crossover comparison of Tc/Cr 24-hour in vivo recovery of 6-week storage in AS-1 versus 12-week storage in EAS-76 variant 6 (EAS-76v6) having 30 mEq per L NaCl. RESULTS: RBCs stored in the lower salt variants of EAS-76 had higher concentrations of RBC ATP with less hemolysis and microvesiculation. RBC 2,3 DPG was preserved for two weeks. RBCs stored for 12 weeks in EAS-76v6 exhibited 78 +/- 4 percent 24-hour in vivo recovery. CONCLUSIONS: It is possible to store RBCs for 12 weeks with acceptable recovery and 0.6 percent hemolysis and with normal 2,3 DPG concentrations for 2 weeks.     

The effect of hypotonicity, glutamine, and glycine on red cell preservation

BACKGROUND : Red cells (RBCs) stored in hypo-os-molar additive solutions with the same concentrations of adenine, dextrose, mannitol, and sodium chloride and varied amounts of ammonium, phosphate, glycerol, and glutamine were better preserved than RBCs in the standard additive solution (Adsol). Cell swelling occurred in all the experimental additives. This observation prompted the evaluation of glutamine and glycine alone, as well as a combination of glutamine and glycine, all of which have been described as producing swelling of rat liver cells. STUDY DESIGN AND METHODS : Aliquots of RBCs were stored at 4°C in Adsol or experimental additive solutions (EASs) all containing adenine, 2 mM; dextrose, 110 mM; mannitol, 55 mM; and sodium chloride, 50 mM. EAS 42 had, in addition, glutamine, 10 mM; glycine 5 mM; and phosphate, 20 mM. EAS 43 had glutamine, 10 mM; glycine, 10 mM; and phosphate 20 mM. EAS 44 had glutamine, 10 mM; EAS 45 had glutamine, 10 mM, and phosphate, 20 mM; and EAS 46 had only glycine, 10 mM. At intervals, measurements were made of mean corpuscular volume, mean corpuscular hemoglobin concentration, morphology, ATP, hemolysis, supernatant potassium, ammonia, pH, and microvesicles shed. RESULTS : The initial mean corpuscular volumes were larger in all EASs than in Adsol, but the greatest difference was between EASs 44 and 46 (108 fL) and Adsol (86 fL) (p<0.001). The morphology scores were significantly better in all the EASs (p<0.04). The ATPs were significantly greater in all the EASs (p<0.001), and highest in those with phosphate. Potassium leakage and hemolysis were less in the EASs (p<0.001). The ammonia levels were higher in all the EASs than in Adsol, with the exception of EAS 46. During storage, the extracorpuscular and intracorpuscular pH levels were essentially identical. The shedding of microvesicles was greatly reduced in all the EASs. CONCLUSION : Cell swelling induced in RBCs after collection appears to improve preservation. Ammonia and phosphate enhance RBC ATP maintenance. Glycine decreases the formation of ammonia by RBCs stored in a hypotonic medium.     

Inactivation of WBCs in RBC suspensions by photoactive phenothiazine dyes: comparison of dimethylmethylene blue and MB

BACKGROUND: The transfusion of blood components containing WBCs can cause unwanted complications, which include virus transmission, transfusion-associated GVHD, alloimmunization, febrile reactions, and immunomodulation. Phototreatment with 4 microM of dimethylmethylene blue (DMMB) and 13 J per cm(2) of white light irradiation has previously been shown to be an effective way to inactivate different models of enveloped and nonenveloped viruses in RBC suspensions, with minimum damage to RBCs. The present study compares WBC photoinactivation in buffy coat after DMMB or MB phototreatment under virucidal conditions. STUDY DESIGN AND METHODS: Buffy coat diluted to 30-percent Hct was treated with the dye and white light. Isolated WBCs were assayed for cell proliferation and viability by an assay using a tetrazolium compound, limiting dilution analysis, DNA fragmentation, and flow cytometry assays. RESULTS: DMMB and 2.5 J per cm(2) of light phototreatment can inactivate T cells to the limit of detection by limiting dilution analysis (>4.76 log reduction). No WBC proliferation activity was observed after DMMB and 3.8 J per cm(2) of light. DNA degradation after DMMB phototreatment was light dependent. In addition, DMMB phototreatment induced apoptosis in WBCs. In contrast, MB phototreatment under virucidal conditions did not cause significant changes in the viability of WBCs. Neither DNA degradation nor signs of apoptosis were observed after MB phototreatment. CONCLUSION: DMMB phototreatment inactivates T-lymphocytes, the cells that cause GVHD.     

Successful storage of RBCs for 9 weeks in a new additive solution

BACKGROUND: This study explored the effect of storing packed RBCs suspended in 200 mL of an alkaline, hypotonic, experimental additive solution (EAS 61). STUDY DESIGN AND METHODS: Packed RBC units prepared from RBCs collected from healthy donors in CPD were stored for 8 (n = 10) and 9 (n = 10) weeks under blood bank conditions after the addition of 200 mL of EAS 61 (adenine, 2 mM:; dextrose, 110 mM:; mannitol, 55 mM:; NaCl, 26 mM:; Na(2)HPO(4), 12 mM:). Standard methods were used for in vitro assays. The 24-hour in vivo autologous recoveries were measured with (51)Cr. RESULTS: Mean +/- SD recoveries at 8 and 9 weeks were 81 +/- 7 and 77 +/- 7 percent. After 9 weeks, the ATP of the RBCs was 81 percent of the initial value, hemolysis was 0.35 percent, supernatant potassium was 46 mEq per L, and the morphologic index was 94.1. CONCLUSION: Packed RBCs suspended in 200 mL of EAS 61 can be stored satisfactorily for 9 weeks. Longer RBC storage should reduce outdating, increase availability of transfusions in remote locations, and improve the efficiency of autologous donor programs.     

Successful storage of RBCs for 10 weeks in a new additive solution

BACKGROUND: The effect of storing packed RBCs suspended in 300 mL of an alkaline, experimental additive solution (EAS 64) was explored. STUDY DESIGN AND METHODS: RBC units prepared from blood collected from healthy donors into CPD were WBC reduced and stored for 10 weeks under blood bank conditions after the addition of 300 mL of EAS 64 (adenine, 2 mM:; dextrose, 50 mM:; mannitol, 20 mM:; NaCl, 75 mM:; Na(2)HPO(4), 9 mM:). For comparison, non-WBC-reduced units from the same donors were stored in a different additive solution (AS-1, Baxter Healthcare) for 6 weeks. Standard methods were used for the in vitro assays. The 24-hour in vivo recoveries were measured by using (51)Cr- and (99m)Tc-labeled RBCs. RESULTS: Mean recovery in the EAS 64 units after 10 weeks was 84 +/- 8 percent, the same as in the AS-1 units stored for 6 weeks. For EAS 64 and AS-1 units, respectively, the ATP of the RBCs was 85 percent and 64 percent of the initial value, hemolysis was 0.43 percent and 0.63 percent, supernatant potassium was 24 mEq per L and 44 mEq per L, and the morphologic index was 98 and 71. CONCLUSION: RBCs suspended in 300 mL of EAS 64 can be stored satisfactorily for 10 weeks. Longer RBC storage should reduce outdating, increase availability of transfusions in remote locations, and improve the efficiency of autologous donor programs.     

Buffering and dilution in red blood cell storage

BACKGROUND: Red blood cell (RBC) storage solutions work in a narrow pH range between 7.2 and 6.4. While keeping RBC within that pH range, ATP production can be increased by buffering or dilution. STUDY DESIGN AND METHODS: In the first study, 12 units of packed CP2D RBCs were pooled in groups of four, re-aliquoted, and added to one of four additive solutions (ASs): AS-3, 110 mL; EAS-61, 170 mL; EAS-78, 170 mL; or EAS-81, 110 mL. EAS-78 and -81 contain bicarbonate. Units were sampled approximately weekly for 10 weeks for biochemical measures. In the second study, 12 volunteers donated RBCs for measures of (51)Cr in vivo recovery after 6 or 8 weeks of storage in EAS-81. RESULTS: RBCs stored in the higher-volume or buffered ASs had higher RBC ATP concentrations. The combination had an additive effect. Hemolysis was reduced in dilute ASs and less so with buffering. RBCs stored for 8 weeks (n=6) in EAS-81 exhibited 87+/- 2 percent 24-hour (51)Cr in vivo recovery and 0.4+/- 0.2 percent hemolysis. CONCLUSIONS: It is possible to store RBCs for 8 weeks in buffered conventional volume ASs. Combining buffering and increased AS volume improves stored RBC characteristics further.     

Prolonged maintenance of 2,3-diphosphoglycerate acid and adenosine triphosphate in red blood cells during storage

BACKGROUND: Current additive solutions (ASs) for red cells (RBCs) do not maintain a constant level of critical metabolites such as adenosine triphosphate (ATP) and 2,3-diphosphoglycerate acid (2,3-DPG) during cold storage. From the literature it is known that the intracellular pH is an important determinant of RBC metabolism. Therefore, a new, alkaline, AS was developed with the aim to allow cold storage of RBCs with stable product characteristics. STUDY DESIGN AND METHODS: Whole blood-derived RBCs (leukoreduced) were resuspended in experimental medium phosphate-adenine-guanosine-glucose-gluconate-mannitol (PAGGG-M; pH 8.2) with and without washing in the same medium. During cold storage several in vitro variables, such as intracellular pH, 2,3-DPG, ATP, and hemolysis, were analyzed. RESULTS: During cold storage, RBCs resuspended in PAGGG-M showed a constant ATP level (approx. 6 mumol/g Hb) and a very limited hemolysis (<0.2%). The 2,3-DPG content showed an increase until Day 21 (150% of initial level), followed by a slow decrease, with at Day 35 still 100 percent of the initial level. RBCs washed in PAGGG-M even showed a continuous increase of 2,3-DPG during 35 days, with a maximum level of 200 percent of the initial value. The effect of PAGGG-M appears to be related to long-lasting effects of the initial intracellular pH shortly after production. CONCLUSION: Resuspension of RBCs in our alkaline medium PAGGG-M resulted in a RBC unit of high quality during storage for up to at least 35 days, with 2,3-DPG levels of higher than 10 mumol per g Hb, hemolysis of less than 0.2 percent, and ATP levels of higher than 5 mumol per g Hb.     

The effect of patient-controlled analgesia on coadministered red blood cells

BACKGROUND: Patient-controlled analgesia (PCA) provides effective pain control. The possibility of administrating opioids in the same line as red blood cells (RBCs) for patients with poor venous access has been entertained. The literature on this approach is not extensive, but generally cautionary. STUDY DESIGN AND METHODS: Standard concentrations of morphine, hydromorphone (Dilaudid), and meperidine (Demerol) were used to determine the effect on RBCs. Three in vitro approaches were used: 1) continuous low-dose opioid infusion with a single bolus, 2) continuous infusion with multiple boluses, and 3) assessment of RBCs with different concentrations of opioids in test tubes. Samples were assayed for hemoglobin (Hb), mean corpuscular volume (MCV), plasma Hb, potassium, and lactate dehydrogenase, and a peripheral blood smear was made. RESULTS: Addition of each drug as a single or multiple bolus(-es) with continuous infusion showed the same effects as normal saline. In vitro exposure of Demerol at a 1:2 ratio (drug:blood) increased the MCV (110 fL), at 1:1 the MCV was 120 fL, and there was 4.5 percent hemolysis. At 2:1, hemolysis increased to 9.2 percent. Both morphine and Dilaudid had similar effects as normal saline. CONCLUSION: Morphine, Dilaudid, and Demerol, given as a bolus in the intravenous line, have the same effects as those seen with saline. When mixed directly with the blood for more than 1 hour, however, Demerol caused increasing RBC swelling and at high, nontherapeutic concentrations, caused hemolysis. Our study suggests that analgesia delivered via PCA may be safely coadministered with RBCs. Further clinical study is warranted.     

Evaluation of a concurrent multicomponent collection system for the collection and storage of WBC-reduced RBC apheresis concentrates

BACKGROUND: Multicomponent apheresis procedures offer the possibility of collecting blood components that are standardized, as compared to those available with whole-blood donations. A new separator program for the concurrent collection of RBCs, platelets, and plasma (Amicus, Baxter Healthcare) was evaluated. STUDY DESIGN AND METHODS: Apheresis donors (n = 47) underwent concurrent collection of RBCs, platelets, and plasma by use of the single-needle procedure of the Amicus blood cell separator. A standardized RBC volume (100% Hct) of 200 mL was targeted with either 1 or 2 platelet concentrate units, depending on the donor's predonation characteristics. After collection, the RBC component was sterilely connected to an RBC collection set (Amicus) to allow for the addition of 100 mL of saline-adenine-glucose-mannitol preservative solution and WBC reduction at either ambient temperature or 4 degrees C. The RBC units were subsequently stored at 2 to 6 degrees C for 42 days, and the following in vitro measures were evaluated over the storage period: blood cell counts including Hct and total Hb, plasma Hb, potassium, pH, ATP, and 2,3 DPG. RESULTS: Procedure time averaged 74 +/- 9 minutes, and no adverse events were reported. The absolute RBC volume collected averaged 198 +/- 11 mL with an average Hct value of 83 +/- 2 percent. After filtration, the Hb content averaged 58.2 +/- 2.4 g per unit and residual WBCs averaged 0.038 +/- 0.015 x 10(6) per unit. Day 42 results showed that all units had on average more than 70-percent ATP maintenance, and all of the units had less than 0.8 percent he-molysis. All units had pH values higher than 6.5 on Day 42. CONCLUSION: The concurrent multicomponent collection system (Amicus) can reliably collect a standardized RBC unit of good quality. In vitro testing of the RBCs collected and stored for 42 days met the Council of Europe criteria for transfusion.     

Virus inactivation in red cell concentrates by photosensitization with phthalocyanines: protection of red cells but not of vesicular stomatitis virus with a water-soluble analogue of vitamin E

BACKGROUND: Photodynamic treatment of red cells (RBCs) with phthalocyanines and red light inactivates lipid-enveloped viruses, such as vesicular stomatitis virus (VSV) and human immunodeficiency virus. To protect RBCs from photodynamic damage, type I free radical quenchers, such as mannitol, which did not affect virus inactivation, were added. STUDY DESIGN AND METHODS: Aluminum phthalocyanine tetrasulfonate (AIPcS4) was found to inactivate VSV at a rate one- fourth that of the silicon phthalocyanines (Pc 4 and Pc 5). However, the latter also caused more RBC damage. To protect RBCs against this photodynamic damage, Trolox, a water-soluble vitamin E analogue, was used. RBC damage was measured as potassium leakage or hemolysis during storage after treatment. In addition, reduction in negative surface charge on RBCs was measured immediately after treatment, and the effect of Trolox on VSV inactivation in RBCs was evaluated. RESULTS: Trolox at a concentration of 5 mM was found to reduce potassium leakage during storage after Pc 4 and AIPcS4 photodynamic treatment of RBCs. Hemolysis during storage of RBC concentrates treated with Pc 4 or Pc 5 was drastically reduced by the addition of 5 mM Trolox prior to light exposure. At the same concentration, Trolox inhibited the reduction of negative surface charges on RBCs following Pc 4 and Pc 5 photodynamic treatment. Under these conditions, VSV inactivation by photodynamic treatment with all phthalocyanines was not affected by Trolox. In aqueous solution, Trolox formed a complex with AIPcS4, thus quenching the excited triplet state of AIPcS4 at a constant rate of 8.8 × 10(6) per M per second. CONCLUSION: These findings indicate that Trolox protects RBCs from phthalocyanine-photosensitized damage without affecting virus kill. The addition of Trolox would be beneficial for improving the quality of RBCs subjected to photodynamic treatment.     

Storage of red blood cells with improved maintenance of 2,3-bisphosphoglycerate

BACKGROUND: During storage, red blood cells (RBCs) rapidly lose 2,3-bisphosphoglycerate (2,3-DPG) leading to an increase in the affinity for O(2) and a temporary impairment of O(2) transport. Recent clinical evaluations indicate that the quality of transfused RBCs may be more important for patient survival than previously recognized. STUDY DESIGN AND METHODS: Glucose-free additive solutions (ASs) were prepared with sodium citrate, sodium gluconate, adenine, mannitol, and phosphates at high pH, a solution that can be heat-sterilized. CP2D was used as an anticoagulant. Additional CP2D was added to the AS to supply glucose. RBCs were stored at 4 degrees C and assayed periodically for intracellular pH (pHi), extracellular pH, glucose, lactate, phosphate, ATP, 2,3-DPG, hemolysis, and morphology. RESULTS: Storage in 175 mL of the chloride-free, hypotonic medium at a hematocrit (Hct) level of 59 to 60 percent resulted in an elevated pHi and the maintenance of 2,3-DPG at or above the initial value for 2 weeks without loss of ATP. The addition of 400 mL of storage solution followed by centrifugation and removal of 300 mL of excess solution to a Hct level of 60 to 66 percent further reduced the chloride concentration, resulting in the maintenance of 2,3-DPG for 4 weeks. Hemolysis was at 0.1 percent at 6 weeks. CONCLUSION: Improvements in the maintenance of 2,3-DPG were achieved with 175 mL of a chloride-free storage solution with familiar additives at nontoxic concentrations to increase pHi. Adding, instead, 400 mL of storage solution followed by the removal of 300 mL reduced the chloride concentration, increasing the pHi and extending the maintenance of 2,3-DPG to 4 weeks.