Prolonged storage of red cells at 4 degrees C

One of the events associated with red cell storage at 4 degrees C is the development of an increasing proportion of echinocytes. Vesicles also may bud off the spicules, presumably leading to a decreased surface-to-volume ratio and decreased deformability. Pursuing the hypothesis that increasing the surface tension of the cells by increasing their volume might reduce the tendency toward echinocytosis and extend refrigerated storage time, packed red cells were resuspended in a solution hypotonic (210 mOsm) with respect to solutes that do not penetrate the cell. Since a reduced ionic concentration results in increased membrane permeability for cations, normal ionic concentration was maintained by the addition of NH4C1, which readily penetrates red cells and therefore contributes no osmotic support. Adenine, glucose, mannitol, citrate, and phosphate also were included. Unexpectedly, the predominant effect of red cell storage in this solution was a remarkable elevation of adenosine triphosphate (ATP). At 4, 8, and 10 weeks, (ATP) levels averaged 165, 135, and 110 percent of initial values, respectively. At 16 weeks, ATP still averaged 50 percent of initial values. Twenty-four-hour in vivo survival of red cells measured at 12 to 18 weeks ranged between 70 and 80 percent, and hemolysis ranged from 0.3 to 7.1 percent. Both the hypotonicity and the ammonium salt appear to be necessary for the high ATP.     

Liquid storage at 4 degrees C of previously frozen red cells

Fresh human blood was collected in citrate-phosphate-dextrose, frozen by a high-glycerol technique, and stored at -80 degrees C. The red cells were thawed, deglycerolized, and resuspended in a final wash solution, ADSOL (Fenwal Laboratories), or an additive solution (AS) containing glucose, adenine, mannitol, and phosphate. The cells were then stored at 4 to 6 degrees C for 21 days and assayed weekly for adenosine triphosphate and 2,3 diphosphoglycerate, pH, glucose use, and lysis. AS and, to a lesser extent, ADSOL produced metabolic profiles similar to or better than profiles of cells not frozen and stored in commercially available additive solutions. AS offers a potential post-thaw preservative solution for red cells that would greatly increase the flexibility and reduce the expense of using frozen blood. A sterile post-thaw storage capability will make the stockpiling of frozen red cells a practical concept for both military and civilian blood banks.     

Human lactoferrin induces apoptosis-like cell death in Candida albicans: critical role of K+-channel-mediated K+ efflux

Human lactoferrin (hLf) induced an apoptosis-like phenotype in Candida albicans cells, which includes phosphatidylserine externalization, nuclear chromatin condensation, DNA degradation, and increased reactive oxygen species (ROS) production. Intracellular ROS accumulation was seen to correlate with candidacidal activity in hLf-treated cells. Mitochondrial activity was involved as indicated by mitochondrial depolarization and increased hLf resistance of cells preincubated with sordarin or erythromycin, the latter of which inhibits protein synthesis in mitoribosomes. Interestingly, Cl⁻- and K⁺-channel blockers prevented the hLf antimicrobial activity, but only when cells were pretreated with the blocking agent (tetraethylammonium) prior to the hLf-induced K⁺-release period. These results indicate for the first time that K⁺-channel-mediated K⁺ efflux is required for the progression of apoptosis-like process in yeast, suggesting that this essential apoptotic event of higher eukaryotes has been evolutionary conserved among species ranging from yeasts to humans.     

Stability of imipenem in Mueller-Hinton agar stored at 4 degrees C.

The purpose of the present study was to measure the stability of imipenem in Mueller-Hinton agar stored at 4 degrees C over time. MICs for Staphylococcus aureus ATCC 25923, Streptococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 were determined in triplicate daily for up to 15 days. The calculated mean time to observe a shift of one dilution in MIC endpoints was 4.33 +/- 1.25 days. For routine work, imipenem agar dilution plates should be prepared within 48 to 72 h of the test.     

Morphological classification by scanning electron microscopy of erythrocytes stored at 4 degrees C in citrate-phosphate-dextrose anticoagulant

Scanning electron microscopy of human blood stored at 4 degrees C in CPD buffer shows a variation of red cell morphology over a period of 21 days. Our results show that at least 10 significantly different red cell shapes are present in stored whole blood.     

Short-term skin preservation at 4 degrees C: skin storage configuration and tissue-to-volume medium ratio

This study was designed to examine the effect of the storage configuration of skin and the ratio of tissue-to-storage medium on the viability of skin stored under refrigeration. Human skin specimens were stored in four physical configurations in RPMI 1640 tissue culture media at 4 degrees C. Skin was transferred to surgically created defects on nude mice after specific storage intervals. Grafts were examined grossly and microscopically after ten days. In the rolled configuration, on storage day 15, the viability of the outside of the roll was significantly better than the inside (P less than 0.01). The graft viability of the outside of the skin rolls was similar for both tissue-to-media ratios as well as for both free-floating configurations (P = 0.27). These findings suggest the optimum cold storage configuration is free floating, and 300 cm2/100 mL is an appropriate skin surface area to volume media ratio. This proportion of tissue to media is in agreement with the minimum ratio currently recommended by the Skin Council of the American Association of Tissue Banks.     

4-Hydroxylation of debrisoquine by human CYP1A1 and its inhibition by quinidine and quinine

A panel of 15 recombinant cytochromes P450 expressed in human B-lymphoblastoid cells was used to study debrisoquine 4-hydroxylation. Both CYP2D6 and CYP1A1 carried out the reaction. The apparent K(m) (micromolar) and V(max) (picomoles per minute per picomole of P450) for CYP2D6 were 12.1 and 18.2 and for CYP1A1 were 23.1 and 15.2, respectively. CYP1A1 debrisoquine 4-hydroxylase was inhibited by the CYP1A1 inhibitor alpha-naphthoflavone and the CYP1A1 substrate 7-ethoxyresorufin. Additionally and surprisingly, this reaction was also inhibited by quinidine and quinine, with respective IC(50) values of 1.38 +/- 0.10 and 3.31 +/- 0.14 microM, compared with those for CYP2D6 debrisoquine 4-hydroxylase of 0.018 +/- 0.05 and 3.75 +/- 2.07 microM, respectively. Anti-CYP1A1 monoclonal antibody (mAb) 1-7-1 abolished CYP1A1 debrisoquine hydroxylase and anti-CYP2D6 mAb 50-1-3 eradicated CYP2D6 debrisoquine 4-hydroxylase. Three further CYP2D6-specific reactions were tested: dextromethorphan O-demethylation, bufuralol 1'-hydroxylation, and sparteine dehydrogenation. The CYP2D6 specificity, judged by the CYP2D6/CYP1A1 activity ratios was 18.5, 7.0, 6.0, and 1.6 for dextromethorphan, bufuralol, sparteine, and debrisoquine, respectively. Thus, debrisoquine is not a specific CYP2D6 substrate and quinidine is not a specific CYP2D6 inhibitor. These findings have significant implications for the conduct of in vitro drug metabolism inhibition studies and underscore the fallacy of "specific chemical inhibitors" of a supergene family of enzymes that have overlapping substrate specificities. The use of highly specific mAbs in such studies is mandated. It is unclear as yet whether these findings have implications for the relationship between CYP2D6 genotype and in vivo debrisoquine 4-hydroxylase activity.     

Contractile properties of rat skeletal muscles following storage at 4 degrees C.

The purpose of this study was to assess the potential of preservation solutions for protecting skeletal muscle function during storage at 4 degrees C. The soleus and the cutaneus trunci (CT) from the rat were stored for 2, 8 or 16 h at 4 degrees C in University of Wisconsin solution (UW), HTK-Bretschneider solution (HTK) or Krebs-Henseleit solution (KH). After storage, muscles were stimulated electrically to analyse the isometric contractile properties, such as the maximum tetanic tension (P(0)). Histological analysis was also performed. In separate experiments, the effect of the diffusion distance on muscle preservation was studied by bisecting the soleus. After 8 h of storage in UW or HTK, the contractile properties of the CT were similar to those of the control, whereas those of the soleus were reduced (P(0) values of 16% and 69% of control in UW and HTK respectively). At 16 h, the contractile properties of the CT (P(O) 28%) were again better preserved than those of the soleus (P(0) 9%). Muscle function deteriorated most after storage in KH (P(0) at 16 h: soleus, 3%; CT, 17%). The bisected soleus was equally well preserved as the CT (P(O) of bisected soleus at 8 h in UW and HTK: 86%). The functional data corresponded well with the histological data, which showed increasing muscle fibre derangement with increasing storage time. For both muscles and all solutions, the threshold stimulus current increased with increasing storage time (control, 0.1 mA; 16 h, 1.2 mA) and was strongly correlated with the deterioration in contractile properties. It is concluded that, at 4 degrees C, muscle is preserved better in UW and HTK (intracellular-like solutions) than in KH (extracellular-like solution). The soleus and CT were best protected in HTK. The diffusion distance is a critical factor for successful preservation of muscle function at 4 degrees C. The reduced function after 16 h of storage at 4 degrees C was caused by hypercontraction and necrosis of about 25% of the muscle fibres, and by deterioration of the electrical component of excitation-contraction coupling of the remaining fibres.     

A comparison of storage viability of nonmeshed and meshed skin at 4 degrees C

Skin stored in nutrient medium at 4 degrees C produces acceptable short-term viability. This study compared the storage viability of nonmeshed v meshed skin stored at 4 degrees C in nutrient medium. Skin specimens from six human donors were stored for up to 35 days in RPMI 1640 tissue culture medium at 4 degrees C. Skin specimens (1 cm in diameter) were transplanted to surgically created defects on the thorax of nude mice at fixed intervals during the storage period. Gross and microscopic techniques were used to determine the graft viability at 10 days postgraft. Skin was divided into two storage groups as nonmeshed or meshed 1.5/1. The storage configuration was free-floating, 10 cm x 2 cm sheet grafts. The ratio of skin surface area to volume medium was 300 cm2/100 mL. There was no significant difference between the viability of the nonmeshed group compared to the meshed group. Prior meshing of human allograft does not adversely affect the viability of banked skin. Therefore, skin can be stored in a meshed configuration. This eliminates operating room time spent preparing allograft for application, which is cost-effective.     

Ca2+-activated K+ efflux limits complement-mediated lysis of human erythrocytes.

The lytic effect of complement on human erythrocytes has been reported by others to increase when Na+ is substituted for K+ in the external medium. In this paper we have investigated the hypothesis that net loss of K+ through a K+ transport pathway protects erythrocytes from complement-induced colloidosmotic swelling and lysis. Antibody-sensitized human erythrocytes containing different intracellular cation concentrations (nystatin treatment) were exposed to low concentrations of guinea pig serum in media of different cation composition; complement lysis was assessed by the release of hemoglobin and the volume of the surviving cells estimated by their density distribution profiles. Complement-dependent swelling and lysis of erythrocytes (a) were limited by the presence of an outwardly directed K+ electrochemical gradient and (b) were enhanced by carbocyanine, a specific inhibitor of the Ca2+-activated K+ transport pathway, and by absence of Ca2+ in the external medium. We propose that during complement activation a rising cytosolic calcium triggers the Ca2+-activated K+ permeability pathway, the Gardos effect, produces a net K+, Cl- and water loss, and thus limits the colloidosmotic swelling and lysis of erythrocytes.     

Effects of the addition of second-messenger effectors to platelet concentrates separated from whole-blood donations and stored at 4 degrees C or -80 degrees C

BACKGROUND: Platelet concentrates (PCs) are currently stored at 22 degrees C under continuous agitation. Because of the potential risk of the overgrowth of bacteria in case of contamination, PC shelf life is limited to 5 days. A mixture of second-messenger effectors is being evaluated to determine if it has benefits for cold liquid storage and cryopreservation of platelets. STUDY DESIGN AND METHODS: PCs separated from whole-blood donations by the buffy coat method were randomly assigned (n = 6 each) to be stored for 5 days at 22 degrees C under continuous agitation or at 4 degrees C after treatment with a platelet storage medium (ThromboSol, LifeCell Corp. ). PCs were also cryopreserved with 6-percent DMSO (final concentration) or with ThromboSol plus 2-percent DMSO (final concentration) (TC). After storage, platelets were analyzed by flow cytometry, transmission electron microscopy, and aggregation and perfusion techniques. RESULTS: Cold liquid storage of ThromboSol-treated platelets resulted in a lower binding of coagulation factor Va on the platelet surface than on platelets stored at 22 degrees C. In transmission electron microscopy, a conversion to spherical morphology was seen in the case of cold liquid storage. No difference between ThromboSol-treated platelets stored at 4 degrees C and platelets stored at 22 degrees C was seen in perfusion studies. Cryopreservation in the presence of TC prevented the reduction in glycoprotein Ib and IV expression on platelet surface that is seen in 6-percent DMSO-cryopreserved platelets. Platelets cryopreserved in TC covered, by thrombus, a significantly greater percentage of the perfused surface after the freezing and thawing process. CONCLUSION: ThromboSol-treated PCs separated from whole-blood donations by the buffy coat method, stored at 4 degrees C for 5 days, or cryopreserved in the presence of TC, maintained in vitro functional activity comparable to that achieved by current methods of storage, although discoid morphology was not preserved during cold liquid storage with ThromboSol.     

Storage of buffy coat-derived platelets in additive solutions: in vitro effects of storage at 4 degrees C

BACKGROUND: The aims of this in vitro study were to compare the storage of platelets (PLTs) at 4 degrees C with those stored at 22 degrees C and to determine the in vitro effects of preincubation at 37 degrees C for 1 hour before the analysis on the basis of the maintenance of PLT metabolic and cellular integrity. STUDY DESIGN AND METHODS: PLT concentrates (PCs) were prepared from pooled buffy coats (BCs) for paired studies (total eight pools from 160 BCs). Each pool was divided into four PCs and stored under different conditions: at 20 to 24 degrees C on a flatbed agitator, at 20 to 24 degrees C on a flatbed agitator and with incubation of the samples at 37 degrees C for 1 hour before the analysis, at 4 degrees C, and at 4 degrees C and with incubation of the samples at 37 degrees C for 1 hour before the analysis. RESULTS: Storage of PLTs at 4 degrees C resulted in reductions in the rate of glycolysis and better retention of pH after Day 10 than in PCs stored at 22 degrees C (Day 14, 7.003 +/- 0.047 vs. 7.201 +/- 0.146). Hypotonic shock response and extent of shape change were higher at 22 degrees C than at 4 degrees C and in preincubated PCs stored at 22 degrees C than in reference PCs stored at the same temperature (Day 5, 45.6 +/- 2.7 vs. 36.5 +/- 3.9 and 24.1 +/- 2.0 vs. 15.5 +/- 1.8). The concentration of RANTES was higher in PCs stored at 22 degrees C than at 4 degrees C (Day 7, 179 +/- 25 vs. 79 +/- 32). CONCLUSION: PLTs stored at 4 degrees C without agitation maintain metabolic and cellular characteristics to a great extent during 21 days of storage. These studies confirm the view that PLTs lose their discoid shape and that this loss with storage at 4 degrees C is associated with reductions in metabolic rate and in their release of alpha-granule content.     

Stability of blood coagulation factors in deep frozen fresh plasma by storage at -20 degrees C and -40 degrees C

Fresh frozen plasma (FFP), was shock-frozen to -25 degrees C within six hours after blood donation. The platelet count was reduced to 20 000/mm3. Aliquots were stored at -20 degrees C and -40 degrees C up to 24 months. Quick, PTT, factor V, VIII, IX, thrombin, antithrombin III, plasminogen, plasma-prekallikrein and kallikrein were determined monthly. With respect to the parameters investigated there was no significant difference between storage at -20 degrees C and -40 degrees C. Factor VIII loss was 10% after 12 months of storage. The activity of factor IX and V remained unchanged during 12 months, then factor V increased during storage. The other parameters did not change. Our study indicates quality of FFP seems not primarily depend on storage temperature, but an optimal preparation technique is much more important.     

The quality of fresh-frozen plasma produced from whole blood stored at 4 degrees C overnight

BACKGROUND: The aim of this study was to assess whether the quality of FFP produced from whole blood stored at 4 degrees C overnight is adequate for its intended purpose. STUDY DESIGN AND METHODS: Fresh-frozen plasma (FFP) separated from whole blood (n = 60) leukodepleted (LD) after storage at 4 degrees C overnight (18-24 hr from donation, Day 1 FFP) was compared with that LD within 8 hours of donation (Day 0 FFP, the current standard method). RESULTS: In more than 95 percent of Day 1 FFP units, levels of factor (F) II, FV, FVII, FVIII, F IX, FX, FXI, and FXII were greater than 0.50 U per mL except for von Willebrand factor (VWF) antigen and FVIII, where 92 and 87 percent of units, respectively, contained greater than 0.50 IU per mL. Compared with historical data on FFP stored for 8 hours, fibrinogen, FV, FVIII, and FXI were reduced by 12, 15, 23, and 7 percent, respectively, but other factors were not significantly reduced. Levels of VWF-cleaving protease activity were not different between FFP prepared from paired units of blood (n = 3) held for 8 or 24 hours, but were below the reference range in an additional 2 of 6 units held for 24 hours. The activities of protein S, protein C, antithrombin III, and alpha(2)-antiplasmin were reduced by less than 10 percent in Day 1 FFP (n = 20), but with final levels above the lower limit of the normal range in greater than 95 percent of units. Activated FXII antigen was not significantly raised in plasma stored for 18 to 24 hours, but levels of prothrombin fragment 1 + 2 were slightly increased (0.88 ng/mL, 18-24 hr; 0.65 ng/mL, < 8 hr). CONCLUSION: These data suggest that there is good retention of relevant coagulation factor activity in plasma produced from whole blood stored at 4 degrees C for 18 to 24 hours and that this would be an acceptable product for most patients requiring FFP.     

Serotonin uptake at 22 degrees C of stored platelets

In order to ascertain the possibility that platelet serotonin uptake may occur during storage of platelet concentrates (PC) at 22 degrees C with agitation, the high-performance liquid chromatographic procedure was applied to determine serotonin uptake by platelets. Studies at 22 degrees C showed that platelets stored for 4 days exhibited a significant serotonin uptake with a Vmax value of 2.4 X 10(-19) mole/platelet/min and a Km value of 0.62 X 10(-6) M. Incubation of PC with 5 X 10(-6) M serotonin for 1 day at 22 degrees C increased their serotonin contents from 2.2 to 4.2 X 10(-7) mole/10(11) platelets. Thrombin stimulation caused about 80% release of intracellular serotonin from fresh as well as stored platelets, which contained standard serotonin in the same amount as the original amount. These results suggest that a significant serotonin uptake of platelets might occur during in vitro storage at 22 degrees C and stored platelets have retained abilities to sequester extracellular serotonin into dense granules.