Intracellular pH in stored erythrocytes. Refinement and further characterisation of the 31P-NMR methylphosphonate procedure.

Methylphosphonate in conjunction with 31P-NMR spectroscopy was used for the measurement of transmembrane delta pH in human erythrocytes stored at 4 degrees C for up to 5 weeks in a nutrient medium. Intra- and extracellular pH was determined using calibration curves based on the pH-dependent separation between the NMR resonances of methylphosphonate and orthophosphate (Pi). A comprehensive statistical procedure is presented for the determination of the variance of NMR-based pH estimates. The entry of methylphosphonate into erythrocytes was more rapid at low pH and uptake was fully inhibited by the band 3 reagent, disodium 4,4-diisothiocyano-2,2'-disulphonic acid stilbene. The distribution ratio of methylphosphonate concentration inside and outside the cells was used to calculate the membrane potential; the analysis depends on a consideration of the Donnan equilibrium for an anion with one or two charges. Furthermore, the analysis does not depend on the pH estimates but relies solely on concentration estimates. The chemical shift of methylphosphonate was not subject to the variations associated with specific intracellular binding encountered with many other phosphorus compounds, including Pi. On the other hand, the ionic strength dependence of the chemical shift of methylphosphonate, contrary to earlier reports, is comparable in magnitude (but opposite in sign) to that of Pi.

The relationship between glucose concentration and rate of lactate production by human erythrocytes in an open perfusion system.

A thermodynamically open system, based on an assembly of capillaries with semi-permeable walls was constructed in order to study glycolysis in human erythrocytes in high haematocrit suspensions. A phenomenological expression for the rate of lactate production as a function of glucose concentration was obtained. The rate was measured under steady-state conditions with low substrate concentrations (approx. 50 mumol/l). In a corresponding closed system, this concentration of glucose would be exhausted within a few minutes. A mathematical model of the whole system consisted of five differential equations, and involved parameters relating to flow rates, volumes of reaction chambers, the rates of lactate efflux from erythrocytes and the expression for the rate of lactate production by red cells. The binding of [14C]pyruvate to haemoglobin and the rate of efflux of [14C]lactate from red cells were measured to yield additional information for the model. The concentrations of ATP and 2,3-bisphosphoglycerate were measured during the perfusion experiments, and a detailed analysis of a model of red cell hexokinase was carried out; the former two compounds inhibit hexokinase and alter the apparent Km and Vmax for glucose in vivo. These steady-state parameters were similar to the glucose concentration at the half-maximal rate of lactate production and the maximal rate, respectively. These findings are consistent with the known high control-strength for hexokinase in glycolysis in human red cells. The practical and theoretical validation of this perfusion system indicates that it will be valuable for NMR-based studies of red cell metabolism using a flow-cell in the spectrometer.

Hemoglobinometry: evaluation of a new method with a stable primary standard.

Using purified chlorohemin as a stable standard, a newly described hemoglobinometry method was evaluated. All blood samples as well as chlorohemin gave identical absorption spectra in an alkaline-detergent solution with a broad peak at 575 nm. Both the reagent mixture and the chlorohemin standard were stable for at least 250 days when stored between 8 degrees C and 32 degrees C in brown glass bottles. The new method showed close correlation when compared to the cyanmethemoglobin technique. Compared to the cyanmethemoglobin method the advantages included a stable standard, shorter conversion time, reduced plasma background and improved hemoglobin ligands conversion. We conclude that the new method confers significant advantages over the cyanmethemoglobin method and in particular provides a stable primary standard with a long shelf-life.

Red cell stroma, a stable standard for assessment of platelet procoagulant release.

Platelet function in vitro may be investigated in various ways. One of the accepted methods involves examination of platelet procoagulant release. Optimal conditions relating to maximal release have not been defined. Red cell stroma phospholipid can substitute for the platelet procoagulant in many in vitro tests (Quick et al., 1954, 1957; Margolis, 1961; Lovric & Margolis, 1964). Using a multiple channel coagulation meter, an assay system was developed for testing both red cell stroma and platelet procoagulants. Maximum procoagulant release was obtained by lysing in alkaline hypotonic solutions or by sonication. The system was tested in a one-stage kaolin-activated platelet-free plasma substrate, obtained from fresh pooled citrated plasma. With this method the amount of available procoagulant could be related to the numbers of red blood cells or platelets. The results indicated that one platelet provided procoagulant equivalent to that derived from 0.7 red cells. Procoagulant release was studied under various conditions and expressed in terms of stable (red cell) standard. Platelets subjected to hypotonic stress or factor 3 release (Hardisty test) were studied quantitatively. The addition of sulphinpyrazone (Anturan) had no observable effect on platelet release in this system.

Supernatant hemoglobin determinations after prolonged blood storage.

Measurement of extracellular hemoglobin is useful for assessing the relative success of different blood storage strategies. The test is now also included in routine quality assurance procedures. With the recent trend towards transfusion of concentrated red cells resuspended in "additive" solutions, a safe and efficient method for the estimation of hemoglobin in these units is required. We have developed a method suitable for this purpose, based on the formation of cyanomethemoglobin. Tetramethylbenzidine (TMB) methods were also investigated and shown to detect hemoglobin and hemoglobin derivatives quantitatively when present in packed cell supernatants. A close correlation was recorded between the estimates of supernatant hemoglobin using the 2 techniques with 68 stored red cell concentrates. Because of its simplicity, adequate sensitivity and avoidance of carcinogenic benzidine derivatives, we recommend the cyanomethemoglobin method for routine use in the measurement of supernatant hemoglobin in stored modified red cell concentrates.

Red cells suspended in a phosphate-fortified additive: biochemical enhancement and longer storage.

When compared to our current storage regime, an additive solution, with 75 mM inorganic phosphate, improved certain biochemical and morphological parameters measured in suspensions of packed red cells throughout 49 days of liquid storage. In particular, the mean ATP level of 20 donations stored with the high phosphate additive was consistently and significantly higher than in standard (n = 6) suspensions. The increased amount of inorganic phosphate and the higher pH of the new additive stimulated ATP production and provided better pH buffering than the standard solution currently in use. Although survival study results indicated adequate 24h survivals for erythrocytes stored for 42 days with the new solution, after 49 days storage, the mean 24h survival of autologous erythrocytes was only 58 +/- 7% (n = 6).

Cryopreservation of rabbit and cat corneas at -18 to -24 degrees C.

A simple method of corneal cryopreservation, in which corneas were frozen at -18 to -24 degrees C, was examined. Rabbit and cat corneas were placed successively in solutions of 50% fetal calf serum in McCarey-Kaufman medium with an increasing glycerol and glucose content. They were then frozen and stored in a -20 degrees C domestic freezer. Rabbit corneas stored in this way were examined in vitro by light and scanning electron microscopy, and both rabbit and cat corneas were also assessed after orthotopic allotransplantation into adult recipient animals. Functional corneal grafts were obtained with rabbit and cat tissue that had been cryopreserved for 3-4 weeks and 1 week, respectively. Endpoint analysis (by light and scanning electron microscopy) of grafts that had survived for 50 days indicated the presence of an intact corneal endothelial monolayer. The corneal endothelium slowly degenerated as the storage time was increased. Importantly, however, the endothelium appeared to withstand the freezing and thawing processes and we conclude that it may be possible to store corneas at temperatures above -196 degrees C, without the need for complex, low-temperature cryopreservation systems.

Alterations in blood components during storage and their clinical significance.

Storage-dependent changes in blood and components as affecting a patient with acute haemorrhage are described. With currently used preservatives and additives blood qualifies as "fresh" for two weeks after collection. Modified red cell concentrates are the optimal form of initial haemotherapy, with the possible exception of massive blood transfusion. Availability of platelet concentrates and other haemostatic blood components as routinely issued by a modern blood bank should meet almost all requirements for an acutely bleeding patient.

Donor blood frozen and stored between -20 degrees C and -25 degrees C with 35-day post-thaw shelf life.

With standard blood bank facilities and interconnected plastic bags donor erythrocytes can be frozen and stored in commercial freezers at temperatures of -20 degrees to -25 degrees C for at least 6 months. The cryoprotectant, a mixture of glycerol and dextrose, is washed off in a closed-circuit system. After being thawed and reconstituted with an additive solution, the pack of red cells can be stored in liquid form for up to 35 days at 4 degrees-6 degrees C. The system lends itself to routine clinical blood transfusion practice.

Packed red cell transfusions -- improved survival, quality and storage.

Various anticoagulant-preservative solutions were investigated with a view to determining which would give optimal shelf life of packed cells. Satisfactory 24-hour post-transfusion survival (greater than 70%) after 28-day storage was obtained with citrate phosphate 277 mM dextrose, 0.25 mM adenine (CP2 X D-adenine). After addition of a rejuvenating electrolyte solution through a multiple closed plastic pack system, there was further improvement in viability, oxygen delivery capacity and metabolic activity of stored erythrocytes, with reduction in both plasma potassium and microaggregate numbers. No untoward reactions were observed during or after transfusion of 440 units of rejuvenated blood.

Improved quality of packed cells.

Improved viability, quality, oxygen delivery capacity and metabolic activity of packed cells was obtained when blood was collected into (CP2D) citrate-phosphate anticoagulant with extra dextrose and, after separation of plasma, stored in an adenine-enhanced electrolyte solution. The system has been incorporated into clinical transfusion practice.

Iron-deficiency anemia: evaluation of compensatory changes.

Compensatory mechanisms in children with iron-deficiency anemia were evaluated by measuring erythrocytic organic phosphates and, in some cases, shifts in the P50 of the oxygen dissociation curve. In 19 children with nutritional anemia (hemoglobin values of 3.2 to 8.2 gm/dl) there was a calculated improved oxygen delivery to tissues equivalent to a hemoglobin level of at least 7.5 gm/dl. Transient decompensation was observed during acidosis. In five children with iron-deficiency anemia due to blood loss and in one child with rheumatoid arthritis no such compensatory changes were observed.

Iron nutrition in infancy: effect of an iron-fortified milk formula.

Iron nutrition in infants was studied in a double-blind trial with artificial milk formula. Statistically significant differences were observed with the iron-fortified formula. Not all haematological indices showed significant differences. The fragiligraph technique result was the only parameter showing differences not influenced by other variables examined.

Iron stores assessed in blood donors by hematofluorometry.

Body iron stores in blood donors were assessed using front-face fluorometric assays of erythrocyte protoporphyrin/heme ratios. Comparison was made with hemoglobin levels and serum ferritin assays. Erythrocyte protoporphyrin/heme assays provide quick and reliable information about possible underlying iron deficiency in the absence of anemia, particularly in regular blood donors.

Flow properties of modified packed red cells.

We investigated free gravitational blood flow of modified packed red cells. Variables affecting free flow included final haematocrit, duration of blood storage at 4-6 degrees C and the centrifugal force required in preparation of this blood component. Irrespective of the duration of storage, free blood flow of modified packed red cells was not different from whole blood, provided the haematocrit did not exceed 0.63.

Regeneration of phosphorylated metabolites in stored erythrocytes in an open perfusion system: studies using 31P NMR spectroscopy.

Human erythrocytes were maintained at high haematocrit in a metabolically functional state for several hours in a thermodynamically open perfusion apparatus. The concentrations of ATP and 2,3-bisphosphoglycerate (2,3-DPG) and pH were continuously monitored before and after metabolic perturbations by using 31P NMR; the monitoring was achieved with a 31P flow-through probe. Methylphosphonate was added to plasma perfusion medium as a phosphorus concentration standard and as a 31P NMR pH probe molecule. The rates of decline of ATP and 2,3-DPG levels in fresh cells in a glucose-free medium were measured as were the rates of reformation in response to a 'rejuvenation' medium. Also, rates of ATP and 2,3-DPG synthesis during perfusion with Krebs bicarbonate-0.5 mmol/l glucose and perfusion with pooled plasma were measured in cells that had been previously stored at 4 degrees C for 5 weeks.

Intra- and extraerythrocyte pH at 37 degrees C and during long term storage at 4 degrees C: 31P NMR measurements and an electrochemical model of the system.

A 31P NMR method based on pH dependent variation of the chemical-shift-difference between the resonances of orthophosphate and methylphosphonate was used to measure simultaneously intracellular pH (pHi) and extracellular pH (pHc) during long term storage of erythrocytes; pH was determined at both 4 degrees C and 37 degrees C. An equation describing the equilibrium distribution of membrane-permeant ions was derived by consideration of the electrochemical and osmotic constraints in the RBC suspension. Calculations using the model-equation and the measured pHi yielded the Donnan ratio and therefore pHo; the relationship between experimentally determined pHi and pHo values was accurately predicted by the model. Sensitivity analysis of the model-equation revealed that the observed increase in transmembrane pH gradient during storage is principally due to the alteration of the total net charge of intracellular (poly)-anions.