Studies of Incomplete Antibodies: I. Effect of Papain on Red Cells

A variety of typical agglutinating G antibodies agglutinated papain-treatedcells in a uniform manner in about 16-fold higher dilutions than nontreatedcells. With M antibodies the corresponding titer increase was on the average4-fold. The papain-treatment of cells also increased the titers of cold agglutinins, phytohemagglutinins and preparations of PVP. Antiglobulin consumption experiments revealed that no more antibody was fixed from the samevolume of antiserum onto papain-treated than onto nontreated cells. Possiblemechanisms underlying enhanced agglutination of enzyme-treated cells werediscussed. It is suggested that the same type of antibody may react with papaintreated and nontreated cells but that a smaller number of antibody moleculesare needed for agglutination of papain-treated cells because of altered surfaceproperties of treated erythrocytes.

Submitted on June 15, 1965 Accepted on September 8, 1965     

Prolonged Storage of Red Cells: The Effect of pH, Adenine and Phosphate

Prolonged storage of red blood cells (RBCs) at 4 °C results in decreased intracellular ATP levels with diminished posttransfusion survival. Meryman described a preservative medium, exceptional in its capacity to increase these intracellular levels during the first weeks of storage and later in maintaining adequate levels, for extended storage periods. We modified this medium, investigated its constituents, and found that its ATP-preserving effect was unrelated to its tonicity or to the presence of mannitol. Throughout storage, RBC potassium leakage and lactate production were moderate. No evidence of osmotic swelling was detected. In spite of high ATP levels, the cells became echinocytes, thus discounting a direct correlation between shape and metabolic status. The most striking finding in this study was that the prestorage pH of the blood unit (pH 7.0), has a crucial contribution in elevating nucleotide levels in a medium containing high levels of phosphate (18–10 m M ) and adenine. We suggest that a combined effect of optimal pH, adenine, glucose and phosphate in the medium contributes to the ability of the RBCs to synthesize the necessary purine nucleotides by the 'salvage pathway'.     

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.     

Quality Assurance and Standards for Red Cells and Platelets

Blood donations vary in composition because of considerable variation in donor cell counts and volume collected. In order to minimize the variation in end products produced, and reduce the likelihood of low quality products, a program called Quality Assurance (QA) is required. Agreement on minimal standards is useful, and a program of quality control (QC) is an important component of QA. However, while QC to ensure compliance with standards is desirable, the objective of manufacturing products of consistently high quality can only be achieved when attention is focused on total process control, self- auditing and the energetic management of errors.     

Perioperative Transfusion Triggers for Red Blood Cells

Perioperative transfusion triggers for red blood cell (RBC) transfusion include physiologic signs of inadequate oxygenation of the entire organism or a specific organ, hemoglobin (Hb) concentration and logistic aspects such as experience of anesthesiologists and surgeons, predictability and magnitude of blood loss and time required for a Hb determination and RBC delivery. At a Hb concentration <6 g/dL a RBC transfusion may be given, however, if the patient is hemodynamically stable one may opt not to transfuse. With Hb concentrations between 6 g/dL and 10 g/dL physiologic signs of inadequate oxygenation decide on RBC transfusion. In absence of hypovolemia signs of inadequate oxygenation include tachycardia and hypotension, an O2 extraction>50%, a a mixed-venous O2 partial pressure <4.3 kPa (32 mmHg), a decrease of O2 consumption >10% (not otherwise explained) and signs of locally deficient oxygenation such as myocardial ischemia. At Hb concentrations >10 g/dL, RBC transfusions are rarely, if ever, indicated.     

Preparation and Preservation of Red Cells

Background : The production of blood components has undergone several changes during the last decades. Methods : Red blood cells will have slightly different properties depending on the way of preparation: whether a hard or soft spin has been used, whether platelets and/or leukocytes have been removed or not, and whether the red cells have been suspended in part of the original plasma or in an additive solution. Automated techniques are now often used for the separation of buffy coats, red cells and plasma. Recently, apheresis techniques have been applied for the preparation of red cells, mostly in combination with plasma or platelets. Continuous addition of the anticoagulant during collection reduces the delay between collection and separation, but the cost is higher and donor time longer. Results : Most of the methods for the preparation and storage of red cells allow 35–42 days of storage with a mean in vivo recovery of >75%. However, the content of erythrocyte 2,3-DPG is commonly lost within 1–2 weeks, caused by the accumulation of acid metabolites, but can be maintained longer with new systems of storage. Leukodepletion of red cells by filtration is used increasingly, but its importance in the majority of transfusions is still unclear. Conclusion : New options for the preparation and storage of red blood cells are available and undergo continuous evaluation     

The Diurnal Rhythm of Enzymes in Human Red Cells

The activities of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP oxidoreductase, G6PD), 6-phosphogluconate dehydrogenase (6-phospho-d-gluconate: NADP oxidoreductase, 6PGD), hexokinase (ATP: D-hexose 6-phosphotransferase, HK), lactic dehydrogenase (L-lactate: NAD oxidoreductase, LDH) and aspirate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, Asp. T) were determined in red blood cells of 11 healthy individuals. The determinations were carried out on samples drawn every 4 h over a 24 h period. The activities of G6PD, 6PGD, LDH and Asp. T exhibited a semi-circadian rhythm, namely, two peaks of activity during 24 h while HK activity demonstrated a true circadian rhythm. In addition a polymorphism of the G6PD and LDH activity patterns was observed. The implications of a biological clock in enucleated cells are discussed.     

Filterability of ACD-Stored Red Cells

The deformability of stored erythrocytes was studied using the Myrenne filtrometer MF4. Blood was stored in ACD under usual blood banking conditions and filterability studies were done at various intervals during a 3-week period. Marked reductions in stored erythrocyte filterability were noted as early as day 5 of storage. These results show that ACD is not a good "rheological' storage medium and indicate that further studies are required, using different storage media to assess the importance and significance of this rheological lesion.     

The Action of Two Sulfhydryl Compounds on Normal Human Red Cells: Relationship to Red Cells of Paroxysmal Nocturnal Hemoglobinuria

Treatment of normal human red cells with AET and cysteine, under suitableexperimental conditions, modifies them in such a way that their behavior inin vitro hemolysis tests becomes similar to that of the erythrocytes of paroxysmal nocturnal hemoglobinuria.

It is felt that alteration of the red cells is due to the -SH groups possessedby both substances. A possible mechanism of action is hypothesized.

Submitted on April 7, 1964 Accepted on June 17, 1964     

The Normal Range of Osmotic Fragility of Red Blood Cells

With the technique of Parpart et al (1947), the normal range for osmotic fragility of red blood cells has been estimated to include 5%–45% haemolysis at a salt concentration corresponding to 4.5 g NaCl/1 (Dacie 1954). This estimate may be questioned, however. Thus, nearly 20% of the data obtained from 50 presumably healthy subjects fell outside these limits. Furthermore, the distribution pattern was very asymmetric with erroneously small standard deviation. On the other hand, if the technical conditions (salt concentration, buffer) were properly adjusted, nearly all the observations were located within the straight part of the s-shaped haemolysis curve and scattered symmetrically around the mean. Under these conditions, the normal range (mean ± 2 SD) included 22%–88% haemolysis. Moreover, this wider range included about 95% of the observations, even adapted to the original experimental situation. The present estimates should therefore replace earlier statements in the literature.     

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.     

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.     

The Intracellular and Membrane Effects of Oxidant Agents on Normal Red Cells

Oxidant drugs produced different patterns of oxidative change in red cell GSH and haemoglobin. Naphthoquinones were associated with a marked increase in methaemoglobin, a marked decrease in GSH and the ready formation of Heinz bodies. Phenylhydrazine, at the lower concentrations, produced Heinz bodies while methaemoglobin levels were only minimally increased and GSH levels unchanged. p -Aminophenol markedly increased the concentration of methaemoglobin while decreasing that of GSH but formed no Heinz bodies. With hydroquinone, GSH was virtually depleted before small amounts of methaemoglobin were produced.
Membrane damage as measured by a loss of red cell K⁺ and haemolysis in hypotonic saline was only found with 1,4-naphthoquinone, β-naphthol and the antimalarial drugs. These compounds produced abnormal osmotic fragility curves which differed one from the other. Membrane injury occurred in the presence and absence of intracellular oxidative change.
These studies demonstrate marked differences in the intracellular and membrane actions of oxidant agents.