Effect of Centrifugation and Subsequent Storage on Red Blood Cells

Blood drawn into CPD solution from 33 normal donors was divided into four groups: (I) centrifuged (at 5,000 g for 7 min) after 7 days of storage, (II) centrifuged after 14 days storage, (III) centrifuged after 21 days storage and (IV) uncentrifuged. After 21 days of storage, aliquots of all units were labeled with chromium-51, reinjected into the donor from which they were drawn and erythrocyte survival was measured. Red blood cell recovery and survival for all four groups was essentially the same; 24-hour recovery was 85%; T 1/2 was 28.2--31.6 days. Our results suggest that blood can be centrifuged and stored at any time during its 21-day shelf life without detrimental effect on erythrocyte survival.     

pH, Temperature and Lactate Production in Human Red Blood Cells: Implications for Blood Storage and Glycolytic Control

The interaction of temperature and pH in biological systems comprises two components. Temperature change may perturb the pH of solutions, and it may change the pKa of some ionizable groups that are involved in enzyme catalysis. The pH optima of single reactions and whole pathways are therefore temperature sensitive. The pH optimum of glycolysis in human red cells has been investigated only at 37 degrees C. We have measured the effect of temperature on the pH of stored blood suspensions and on the pH optimum of glycolysis in the human red cell. The pH of the cell suspensions in a traditional storage medium was 7.25 +/- 0.2 at 4 degrees C. The pH optimum of glycolysis was high (7.8-8.5) between 15 and 35 degrees C. It can be inferred from our data that human red cells are currently stored at least 0.5 pH units below the pH optimum of glycolysis at 4 degrees C. This suggestion is supported by storage experiments which showed that glycolysis at 4 degrees C was at least 1.5-fold more active at an initial pH of 7.67 versus 7.36. Equations which describe the variation in reaction velocity with pH were fitted to the pH curves for glycolysis in order to identify the ionizable groups that contribute to the effect of pH on glycolysis. It is generally accepted that hexokinase catalyses the rate-limiting step in glycolysis in the human red cell, but none of the ionizable groups implicated correspond to that involved in the hexokinase reaction.     

The Effect of o-Salicylate upon Pentose Phosphate Pathway Activity in Normal and G6PD-Deficient Red Cells

The effect of the major metabolite of aspirin, namely salicylic acid, upon the pentose phosphate pathway (PPP) of normal and G6PD-deficient red cells has been studied. Salicylic acid was shown to inhibit this pathway in proportion to the amount present. At any concentration of this substance there was greater inhibition of the PPP in G6PD-deficient than in normal red cells.     

The pH Optima for Papain and Bromelain Treatment of Red Cells

The action of papain and bromelain, prepared over a pH range from 4.6 to 8.6, was evaluated for the ability to render red cells agglutinable by five incomplete antibodies of differing blood group specificities using a two-stage technique. The optimal pH for treatment of red cells by activated papain or bromelain was between 5.4 and 5.8. Above this pH range, a fall in serological sensitivity was apparent which was much more pronounced with papain than with bromelain. The optimal pH for enzyme treatment of red cells can be achieved in two-stage techniques, but not in one-stage techniques due to the buffering effect of serum proteins.     

Preservation of Red Blood Cells: Studies of Erythrocyte Adenine Nucleotides Using Luminescence Analysis

The levels of ATP and total adenine nucleotides (ATP + ADP + AMP) were determined by firefly luciferase assay in red blood cells during storage for 5 weeks at 4 degrees C. With few exceptions, no significant differences in nucleotide levels were found between whole blood stored in CPD-adenine and various preparations of red blood cells in CPD-adenine or CPD with saline-adenine-glucose (SAG) as additive. The levels of ATP and total adenine nucleotides during storage are discussed in relation to glucose levels, extracellular pH and shelf life of the red blood cells.     

Factors Affecting Pentose Phosphate Pathway Activity in Human Red Cells

Summary. The vibrating reed electrometer and ionization chamber have been adapted for the instantaneous and continuous measurement of pentose phosphate pathway (PPP) activity in human red cells. PPP activity was determined by measuring the conversion of glucose-1-¹⁴C to ¹⁴CO₂. PPP activity was independent of ambient glucose concentration over the range of 50–500 mg % and was stimulated up to 36-, 18- and 6-fold by methylene blue, ascorbic acid and primaquine, respectively. PPP activity was increased 24% and 41% when incubation temperatures were raised to 39°C and 40.5°C, respectively. PPP activity in unstimulated RBC was twice as great in 95% O₂ as in 20% O₂ and in 10% O₂ was only slightly less than in 20% O₂. The PPP activity was found to be very pH-dependent. PPP activity in plasma was about twice as great as in Krebs-Ringer bicarbonate buffer. Thus, the activity of the PPP in red cells is altered significantly in vitro by incubation conditions designed to mimic various clinical states.     

Red Cell Preservation: Further Studies with Adenine

Supplementation of the ACD-preservative with small amounts of adenine(0.5 µM per ml. amounting to 37 mg. of the base or 56 mg. of adenine sulfateper 550 ml. unit of blood) preserved satisfactory viability (post-transfusionsurvival greater than 70 per cent) of stored human red cells for 5 to 6 weeks.In the concentrations used, the addition of guanine, cytidine or uridine, aloneor in combination with adenine, had little or no effect in extending viability.Hypoxanthine, even in large amounts, did not appear to be toxic to thestored cells. Preservation of viability after 6 weeks of refrigerated storagemay be somewhat improved by storage in certain plastic containers as compared with glass.

Submitted on April 16, 1962 Accepted on June 22, 1962     

Electrical Impedance Alterations of Red Blood Cells during Storage

The electrical impedance of blood is determined mainly by the resistance of the plasma (R_p), resistance of the red cell interior fluid (R_i), and capacitance of the cell membranes (C_m). These parameters were measured on 10 stored blood samples consecutively during 4 or 5 weeks of storage at 4°C, once every week. Compared to the values of fresh samples, a statistically significant decrease in R_p was found mainly during the first week of storage, R_i did not decrease significantly until after 3 weeks, whereas C_m decreased progressively with time. These alterations can be explained by known red cell lesions during storage. The results indicate that electrical impedance measurements might be useful for monitoring red cell ageing and assessing the quality of stored red blood cells.     

The Effect of Methylene Blue Addition to Whole Blood during Prolonged Storage

Abstract. Addition of methylene blue (MB) in concentrations of 10^–3, 10^–4 and 10^–5 m to CPD blood resulted in lowered levels of glutathione and glutathione reductase. The levels of adenosine triphosphate were not affected by the addition of MB. However, the 2,3-diphosphoglycerate levels remained higher than the control values after 21–42 days of storage at all MB concentrations used. Hemoglobin function was better maintained by the two lower concentrations of MB during most of the storage period. The highest concentration had an adverse effect, lowering the p₅₀ below the control level in CPD.     

Value of Adenine,Various Nucleosides,and Ouabain in Maintenance of Integrity of Red Blood Cells Under Blood Bank Conditions1

Summary. The effects of adenine and various nucleosides on adenine nucleotide metabolism in red cells, as well as on other factors related to the storability of blood were compared during six weeks of storage under blood bank conditions. Adenosine triphosphate (ATP) and total adenine nucleotides were maintained at higher levels, and higher ratios of ATP to adenine diphosphate were observed, in blood containing citrate-phosphate-dextrose (CPD) solution than in blood containing acid-citrate-dextrose (ACD) solution. Higher levels of ATP and total adenine nucleotides were maintained in blood containing ACD together with both adenine and inosine than in blood containing ACD alone or together with only one of the two purines. ACD-adenine was more effective in maintaining adenine nucleotides than was ACD-inosine. Addition of uridine did not have any effect on adenine nucleotides in red cells. ATP and total adenine nucleotides were maintained at higher levels in CPD-adenine than in CPD alone. The effect of CPD-inosine or CPD-adenine-inosine was not investigated. ACD-inosine caused a marked increase in plasma lactate, and inhibited glucose utilization, during storage. An increase in plasma inorganic phosphate was delayed by the addition of inosine, and this effect was augmented by adenine and guanosine. There were no findings suggestive of a beneficial effect of these additives on active cation transport, osmotic fragility, hemolysis, or the deterioration of blood cell morphology. Ouabain, an inhibitor of ATPase, had little effect on red cell nucleotide patterns or on other biochemical or morphological criteria for blood storage, possibly because of the low activity of ATPase at 4°C.     

Additive Solution for the Suspension and Storage of Deglycerolized Red Blood Cells

The additive solution, ADSOL, was evaluated for its suitability in the extended storage of previously frozen, deglycerolized red blood cells. In vitro comparison with red cells suspended in 0.2% dextrose 0.9% saline showed that ADSOL allowed for significantly enhanced adenosine triphosphate preservation throughout the storage period (greater than 2.2 mumol/g Hb beyond 14 days) and for significantly reduced hemolysis (less than 1% beyond 14 days). After 10 days of storage in ADSOL the mean recovery after transfusion was 90% (index of therapeutic effectiveness, 77%). No bacterial contamination was observed. The results suggest that this currently approved additive solution could be used to store red cells for 14 days following thawing, thus avoiding one of the principal drawbacks of frozen red cells.     

The Effect of Age on the Creatine in Red Cells

The creatine in packed red cells was estimated by the diacetyl- i -naphthol technique. The error due to the presence of other guanidino compounds in the cells was found to be small.
The mean value for creatine in the packed red cells of normal females was found to be 5.6 (S.D. ± 1.3) mg. per 100 ml., and in normal males 4.4 (S.D. ± 1.5) mg. per 100 ml. The sex difference is statistically significant.
When red cells were segregated according to age by centrifugation the young red cells contained more creatine than the old cells. The creatine in the mixed population of red cells of patients under treatment for anaemia in whom there was increased erythropoiesis was greatly elevated; values as high as 50 mg. per 100 ml. were observed. With a decline in marrow activity and reticulocytosis there was a persistence of the high red-cell creatine for a long time, suggesting that the loss of creatine from the cells with age is a gradual process.
It is concluded that the level of creatine in the circulating red cells is a sensitive parameter of the mean age of the population.     

Women, Alcohol, and Red Cells

Alcohol abuse is known to increase erythrocyte mean cell volume mainly as a consequence of direct toxic effect on the developing red cell. The influence of alcohol on other red cell parameters is unclear. The objective of this cross-sectional survey was to examine the consequences of different alcohol amounts on red cell parameters among women.
We compared red cell parameters between female alcoholics, heavy drinkers, and controls. Controls ( n = 138) and heavy drinkers ( n = 65) consisted of consecutive 40- and 45-year-old women participating in the health screening, and alcoholics ( n = 73) of consecutive women coming to a detoxification clinic.
Alcoholics had significantly smaller erythrocyte counts ( p < 0.01), and higher erythrocyte mean cell volume values ( p < 0.001), reticulocyte counts ( p < 0.01), and red cell distribution width values ( p < 0.001) than controls. No difference between these groups was found, however, in hemoglobin distribution width value. The only red cell difference between controls and heavy drinkers was erythrocyte mean cell volume, which was significantly higher among heavy drinkers ( p < 0.001). In alcoholics, red cell distribution width values were even more often increased (in 44%) than erythrocyte mean cell volume values (in 34%). This increase in red cell distribution width was not solely explained by iron deficiency or liver disease.
Chronic alcohol abuse not only affects erythrocyte mean cell volume values, but also leads to anisocytosis seen in blood count as an increased red cell distribution width value.     

The Effect of Ficin on the Reaction Between Anti-D and Red Cells

The reaction between a 7S incomplete anti-D and ficin-treated red cells was investigated using ¹³¹I-labelled anti-D. Ficin-treatment brought about an increase in the value of the rate constant for association, and hence an increase in the value of the intrinsic binding constant. There was no evidence from the data that ficin treatment exposes additional antigen sites.

Résumé

La réaction entre un anti-D incomplet 7S et les érythrocytes traités à la ficine a été recherchée en utilisant de l'anti-D marqué au I¹³¹. Le traitement à la ficine assure une augmentation du degré de l'association, et, par conséquent, une augmentation de la constante de liaison intrinsèque. En outre, ces résultats démontrent que le traitement à la ficine n'augmente pas les sites antigèniques.

Zusammenfassung

Mit Hilfe von I¹³¹-markiertem Anti-D wurde die Reaktion zwischen inkompletten Anti-D-Antikürpern vom 7S-Typ und ficinbehandelten Erythrozyten untersucht. Die Ficinbehandlung bewirkte eine Zunahme der Bindungsgeschwindigkeitskonstante mit entsprechender Zunahme der inneren Bindungskonstante. An Hand der Resultate konnte nicht bewiesen werden, daß durch Ficinbehandlung zusätzliche Antigendeterminanten freigelegt werden.     

高葡萄糖刺激血管内皮细胞尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4表达上调、活性氧增加及细胞凋亡

目的研究在高葡萄糖刺激的条件下,人脐静脉内皮细胞中尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4的表达、细胞内活性氧以及细胞凋亡的变化。方法倒置显微镜下观察人脐静脉内皮细胞形态;免疫荧光法检测人脐静脉内皮细胞Ⅷ因子相关抗原的表达;高葡萄糖刺激人脐静脉内皮细胞,用逆转录聚合酶链反应检测尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4mRNA水平,Western blotting检测尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4蛋白表达的变化,DCFH-DA检测细胞内活性氧生成量,流式细胞仪和Hoechst染色检测细胞凋亡。结果高葡萄糖刺激人脐静脉内皮细胞时,尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4mRNA及蛋白表达上调,细胞内活性氧生成增加,细胞凋亡增加。结论高葡萄糖处理促进人脐静脉内皮细胞尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4表达上调并引起细胞内活性氧生成和凋亡增加。     

Prestorage Leukocyte Reduction with In-Line Filtration of Whole Blood: Evaluation of Red Cells and Plasma Storage

Objectives: Prestorage filtration of blood components appears to be an effective method to reduce leukocyte-induced adverse reactions and other complications. To determine whether it is better to filter whole blood before component separation, we compared the efficiency of in-line filtration of whole blood with that of postseparation filtration. Methods: Blood was collected from normal, healthy donors into either regular triple-bag containers or into whole-blood integral-filter container systems. We then compared the in vitro storage values of leukocyte-depleted red blood cell concentrates (RBCC) kept at 4 °C, and plasma frozen for 1 year with nonfiltered blood components as control. Results: All counts of white blood cells after filtration were < 1 × 10⁶ per unit. For almost all storage parameters no significant differences were found between leukocyte-reduced RBCC and control units. The plasma fibrinopeptide A values below 30 ng/ml prior to freezing indicate that filtration does not activate the coagulation factors. Furthermore, the filtration did not influence either the biological values or the coagulation factors of plasma units. Conclusions: Whole blood filtration prior to component preparation seems to offer a useful alternative technique for obtaining leukocyte-reduced RBCC and plasma.     

Changes in Complement Levels and Activity of Red Blood Cells,Fresh Frozen Plasma,and Platelet Concentrates During Storage

Complement cascade plays an important role in the field of transfusion medicine. The study aimed to detect the complement levels of different blood components and different blood types to explore the risk of transfusion of stored blood. The samples including red blood cells (n = 110), fresh frozen plasma (n = 120), and platelet concentrates (n = 104) from healthy blood donors in our center were collected. Complement components (C3, C4, C3b, C3d, and CH50) were assayed to evaluate the activation of complement. The complement levels of various blood components at different storage times were observed. The differences in complement levels of four blood types in various blood components were compared. The complement levels of red blood cells in storage were low, with no significant changes (P > 0.05). C3b and C3d levels in platelets began to significantly increase after storage for 3 days (P < 0.05). The fresh frozen plasma during storage had higher complement levels, and the concentrations of C3 and C4 decreased and C3b and C3d increased at month 4 (P < 0.05). The differences in complement levels of four blood types in various blood components did not significantly change (P > 0.05), but the C3b and C3d levels of AB fresh frozen plasma remained stable during storage, which different from other blood types. The transfusion of red blood cells was relatively safe in terms of complement activation. The activation of complement proteins occurred during the storage of platelet and plasma, except group AB plasma.Electronic supplementary materialThe online version of this article (10.1007/s12288-020-01338-0) contains supplementary material, which is available to authorized users.     

Red Cell Enzyme Activities and Properties of Mutant Pyruvate Kinase after Long-Term Storage of Red Cells in Liquid Nitrogen

After 8 months of storage of normal red cells in liquid nitrogen, red cell enzyme activities were essentially unchanged, except for triose phosphate isomerase which was reduced by about half. Mutant pyruvate kinase (PK), PK Tokyo I, showed no changes in kinetic or electrophoretic properties after 8 months of storage. These findings indicate that red cells stored for a long term in liquid nitrogen can still be used for the study of mutant PK.