Simplified Determination of Blood Adenosine Triphosphate Using the Firefly System

A simplified method is described for the determination of red cell ATPusing the firefly lantern extract method. Variables investigated include theeffect of the time of reading, dilution of firefly extract and the effective rangeof the method. Excellent recoveries were obtained. Optimal extraction of ATPfrom red cells was achieved with a hypotonic buffer at pH 9.2. The methodcould be used with acid-citrate-dextrose, heparin or EDTA as an anticoagulant. The method was found to be highly specific when the nucleotides foundin normal human blood were investigated; only adenosine diphosphate andguanosine triphosphate gave slight readings, neither of which would significantly affect ATP determinations of human blood. Normal human valueswere found to be 5.45 µmoles of ATP/Gm. of hemoglobin or 1.83 µmoles/ml.red cells in heparinized blood samples. This method is believed to be morerapid, more reproducible and more accurate than any previously describedmethod of ATP determination.

Submitted on October 1, 1963 Accepted on December 3, 1963     

Removal of Leucocytes from Whole Blood and Erythrocyte Suspensions By Filtration Through Cotton Wool

Abstract. (1) Blood was stored in polyvinyl-chloride bags containing citrate-phosphate-dextrose (CPD) with adenine in a final concentration of 0.25 mM. (2) Red cell ATP was well maintained (>70% of original) for 4 weeks in whole blood as well as in red cell concentrate (PCV 85 ± 2%). After 5 weeks the ATP level was about 70% in whole blood and about 40% in red cell concentrate. (3) Red cell 2,3-diphosphoglycerate (DPG) was about 60% of the original after 2 weeks and about 30% after 3 weeks of storage when stored both as whole blood and as red cell concentrate. (4) The red cell 24-hour post-transfusion viability was about 80% after 4 weeks of storage both as whole blood and as red cell concentrate. After 5 weeks of storage the 24-hour viability was 78.7 ± 3.5% in whole blood and 76.5 ± 6.7% in red cell concentrate. (5) 820 patients received 3,238 units of CPD-adenine blood, and 761 patients serving as controls received 2,807 units of acid-citrate-dextrose (ACD) blood. The frequency of transfusion reactions was 3.5% for patients receiving CPD-adenine blood and 4.1% for the control group. (6) The maximum storage time was set at 5 weeks for the CPD-adenine blood and 3 weeks for the ACD blood. The longer preservation time decreased out-dating by at least 50%.     

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.     

A New Technic for Separation of Human Leukocytes

A simple technic for separation of human leukocytes is described. It utilizesdifferential sedimentation following red cell and platelet agglutination inducedby Polybrene. In this technic, lymphocytes are partially removed and over50 per cent of polymorphonuclears are recovered. The leukocyte/red cell ratiois about 5, if blood samples with normal leukocyte counts are used. By useof blood samples containing higher white cell counts this ratio may be increased. The separated cells proved to be highly viable as tested by phasemicroscopy, supravital staining and phagocytosis. There was no significantchange in oxygen uptake of leukemic cells in the presence of a relatively largePolybrene concentration.

Accepted on October 30, 1961     

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.     

Red cell stroma in dogs; variations in the stroma protein and lipid fractions related to experimental conditions

Red cell stroma has not been prepared free of iron and/or hemoglobin. It isnot certain whether the iron is a contaminant or an essential constituent of thestroma. Extensive washing does not eliminate iron from the stroma but doescause loss of the stroma constituents, protein and lipid.

Red cell stroma protein is increased in anemia due to blood loss in the dog, onthe average in severe anemia, almost twice the figures recorded in the poolednormal samples of red cell stroma.

Lipid fractions under the same conditions show minor fluctuations: approximately 90 per cent of total lipids in the red cells are recovered in the stroma bythese methods.

The technic for isolation and fractionation of dog erythrocyte stroma is described. Analyses made from the same blood sample by 3 different workers givecomparable results.

Red cell stroma deserves careful study in controlled experimental conditions.

Submitted on October 10, 1952 Accepted on November 26, 1952     

Increased calcium permeability of cold-stored erythrocytes

The calcium, sodium, and magnesium permeability of erythrocytes from blood stored at 4 degrees C in various anticoagulant media has been studied and compared to that of fresh erythrocytes. Passive influx of CA2+ was measured at 37 degrees C in cells pretreated to abolish Ca2+ pumping and was up to fivefold greater for cold-stored erythrocytes than for fresh cells. The Ca2+ leakiness developed gradually after day 2 and reached a maximum by day 7 of cold storage in ACD, CPD, CPD- adenine, or heparin anticoagulants. The total calcium content of cold- stored erythrocytes in ACD was not significantly different from that of fresh erythrocytes. However, when cold-stored erythrocytes were reincubated at 37 degrees C in media containing 1.5 mM ionized calcium and substrates to regenerate ATP, a net gain of Ca2+ occurred that was greater for stored than for fresh erythrocytes. Cold storage of blood for up to 6 wk in any anticoagulant did not alter either sodium or magnesium permeability. Red cell ATP was also measured and fell steadily during cold storage in ACD or CPD, but more increase in Ca2+ permeability preceded any significant change in red cell ATP, it is likely that a selective calcium leak develops independently of the fall in ATP concentration that occurs on cold storage.     

The Production of Hemoglobin C in Sheep Carrying the Gene for Hemoglobin A: Hematologic Aspects

The first part of this study describes the hematologic and physiologicchanges observed in sheep homozygous for the hemoglobin A during severeblood loss anemia. It was found possible thus experimentally to replace Hb Aentirely with a new hemoglobin type, Hb C. The following additional observations were made: (1) Hb C could not be distinguished from Hb A by submitting the appropriate red blood cells to an "acid elution" technic. These twohemoglobin types were found to be more resistant to this treatment than asecond adult hemoglobin type, Hb B, while the fetal hemoglobin of the new-born lamb was found to be highly resistant. In sheep heterozygous for Hb Aand Hb B, both hemoglobin types were equally distributed among all redblood cells. (2) During stages of severe blood loss relatively small quantities ofacid resistant red blood cells of larger size were demonstrable in homozygousHb A sheep; these cells were considered to be reticulocytes. Additional observations regarding variations in red cell parameters are also presented. (3)The oxygen affinities and the Bohr effects of blood samples and red cellhemolysates containing over 90 per cent Hb C are presented and comparedwith those of samples containing only Hb A, Hb B or the hemoglobin of thenewborn lamb.

Attempts to produce Hb C in sheep homozygous for Hb A by means otherthan phlebotomy are described in the second part of this report. Smallamounts of Hb C were demonstrable in a sheep homozygous for Hb A afterrepeated injections of a urinary extract of human origin with high erythropoietic activity. Administration of cobalt and of thyroxin did not result in theformation of significant amounts of Hb C.

Submitted on July 19, 1965 Accepted on December 25, 1965     

Impairment of Red Cell Viability by Exposure to "Excess" Acid--Citrate Dextrose

Collection of blood in "excess" ACD leads to a loss of red cell viability whenthe blood is transfused back into the donor, even without any appreciablestorage period. The mechanism of this loss of viability is not clear. The loss isaccentuated by incubation at 37 C.; it is not affected by varying the dextroseconcentration of the ACD; it cannot entirely be attributed to change in pH ofthe final suspension medium; and it is not related to the degree of swelling ofthe red cells. The loss of viability can completely be corrected by the additionof small amounts of chloride to the ACD.

This effect is presumably the same as the "lesion of collection" described byGibson et al. in relation to viability studies after 28 days of storage.⁴

Submitted on September 27, 1965 Accepted on February 6, 1966     

ATP Metabolism in Pyruvate Kinase Deficient Erythrocytes

Elevated levels of ATP were observed in pyruvate kinase deficient red cellsfrom 5 patients despite increased ATP requirements because of increasedATPase activity. Increased red cell ATPase activity was associated with anexcess of intracellular sodium. Residual aerobic glycolysis in reticulocytes didnot contribute significantly toward ATP generation in these patients’ red cells.Their hemolytic anemia cannot be ascribed to a lack of high energy phosphate.Conversely, hemolysis in vitro appears to require a high energy phosphatedependent metabolic process.

Submitted on January 17, 1967 Accepted on May 15, 1967     

On the Distribution of Red Cell Volumes

A large number of red cell volume distribution curves can be generated onthe same sample of blood by manipulating the length of the Coulter Counteraperture and the shape of the red cell. Curves derived from sphered red cellsflowing through an aperture 70 x 98µ long, or longer appear to be relativelyfree from artefacts and therefore the most likely to be "real."

The use of long apertures to size sphered red cells will give a volume distribution curve that is approximately Gaussian and with a mean that is largerelative to the variance. Whether red cells are Gaussian or log normal in theirvolume distribution is very difficult to determine. On a particle populationwhere volumes are controlled as closely as on red cells (i.e. with such a lowvariance), the normal and the log normal distributions become practicallyindistinguishable. If in the future this decision can be reached by some independent means, then the volume distributions using the Coulter volume transducer can be easily and simply fitted to the shape desired by appropriatemanipulation of aperture length. These ideal volume distribution curves arenot likely to differ markedly from those obtained under the conditions suggested in this paper. For the present, use of a 70 x 98µ aperture to size redcells sphered by suspension in isotonic Sodium Potassium Tartrate will contribute to considerably greater accuracy in identification of red cell subpopulations in abnormal blood.

Submitted on July 28, 1967 Accepted on October 7, 1967     

In vitro Interactions of 51r in Human Red Blood Cells and Hemolysates1,2

Abstract. The intracellular distribution of radioactivity was studied in normal and sickle erythrocytes labeled with sodium ⁵¹ Cr chromate. Both types of cells had a higher fraction of ⁵¹ Cr bound to hemoglobin when labeled in the presence of ACD at a pH of 7.09 than when labeled in the presence of CPD at a pH of 5.96. Citrate at a pH of 5.96 or less entered the red blood cells and decreased the ⁵¹Cr binding hemoglobin. Binding of ⁵¹Cr to hemoglobin within the red cells was also reduced when the ATP and DPG levels in the red blood cells were elevated. Studies with hemoglobin solution showed that ⁵¹Cr binding to hemoglobin was influenced by 2,3-DPG, ATP and citrate.     

Post-thaw storage at 4 degrees C of previously frozen red cells with retention of 2,3-DPG

Fresh human blood was collected in CPD, frozen by either the Meryman or the Valeri high glycerol technique, and stored at -80 degrees C. Later the red cells were thawed, deglycerolized by the appropriate technique and resuspended in either saline-glucose wash solution or an additive solution containing ascorbate-2-phosphate, adenine, glucose (dextrose), mannitol and sodium phosphate. The cells were stored at 4-6 degrees C for 21 days and assayed weekly for ATP, 2,3-DPG, pH, P50, glucose utilization and lysis. The additive solution maintained red cell 2,3-DPG at fresh blood levels for 3 weeks and maintained ATP levels sufficiently well to suggest good red cell viability for 21 days. There was no difference in results between the Meryman or the Valeri freezing methods if sodium phosphate was used with the saline-glucose wash solution in the Valeri method. If this additive solution is coupled with sterile deglycerolization techniques, 3 weeks of post-thaw red cell preservation would be practical. Using this additive solution would make frozen blood a reasonable source of red cells for emergency needs in both military and civilian blood banking.     

A soluble adenosine triphosphate-dependent proteolytic system in human peripheral red blood cells

A soluble adenosine triphosphate (ATP)-dependent proteolytic system has been detected in human peripheral blood erythroid cells. Hemolysates prepared from reticulocyte-rich blood of subjects with autoimmune hemolytic anemia, treated pernicious anemia, and iron deficiency anemia or from pools of red blood cells enriched for reticulocytes by density gradient centrifugation were tested against a radioactive casein standard. Up to 57% of the casein was rendered trichloroacetic acid (TCA) soluble after incubation with such hemolysates for 60 minutes in the presence of 1.0 mmol/L ATP. In the absence of ATP or in hemolysates prepared from reticulocyte-poor blood as little as 6% to 10% of the casein was hydrolyzed. The proteolytic activity was found in the 100,000-g supernatant of active hemolysates and was blocked by hemin, N- ethylmaleimide, and sodium vanadate and thus resembles a previously described activity in rabbit reticulocytes. In the presence of ATP, similar lysates prepared from rabbit reticulocytes preferentially hydrolyzed the abnormal human hemoglobins Leiden and Gun Hill compared with hemoglobin A. These results suggest that there is an active ATP- dependent proteolytic system in young human erythroid cells that can degrade certain abnormal globin chains; the enzymatic activity is lost in the transition from reticulocyte to erythrocyte.     

Extraction and analytic procedures for cytosine arabinoside and 1-beta-D-arabinofuranosyluracil and their 5'-mono-, di-, and tri-phosphates.

A qualitative and quantitative comparison of perchloric acid (PCA) and trichloroacetic acid (TCA) extraction procedures of biologic material containing cytosine arabinoside (Ara-C) and 1-beta-D-arabinofuranosyluracil (Ara-U) and their biologic 5'-phosphate derivatives revealed definite superiority of the PCA procedure over the TCA procedure. High-pressure liquid chromatography provides rapid and accurate quantitative and qualitative separation of both Ara-C and Ara-U and their 5'-mono-, di-, and tri-phosphates (mu Bondapak NH2 column), or their 5'-mono-, di-, and tri-phosphates without nucleosides (ABX Permaphase columns).     

In vivo regeneration of red cell 2, 3-diphosphoglycerate following transfusion of DPG-depleted AS-1, AS-3 and CPDA-1 red cells

Regeneration of 2,3-diphosphoglycerate (DPG) was determined following transfusion of DPG-depleted group O red cells into group A recipients. Blood from five donors was stored in the adenine-containing solutions CPDA-1, AS-1 or AS-3 for 35 d at 4 degrees C. Post-transfusion red cell DPG and ATP were measured in separated group O red cells over a 7 d period. The studies confirmed rapid in vivo DPG regeneration with greater than or equal to 50% of the maximum level being achieved within 7 h. An average of 95% of the recipients' pre-transfusion DPG level was achieved by 72 h and by 7 d mean (+/- SEM) DPG levels relative to recipient's pre-transfusion DPG averaged 84% (+/- 13%), 92% (+/- 17%) and 84% (+/- 21%) for CPDA-1, AS-1 and AS-3 red cells, respectively. Results were comparable to those previously reported for blood stored in ACD for 15-20 d (Valeri & Hirsch, 1969; Beutler & Wood, 1969). The immediate regeneration rate, V, closely approximated first order regeneration kinetics with AS-3 red cells exhibiting double the rate of CPDA-1 red cells (P less than 0.001). AS-1 red cells exhibited an intermediate rate of regeneration which was not significantly different compared to either CPDA-1 or AS-3 (P greater than 0.05). V exhibited a significant (P less than 0.05) positive correlation with ATP levels 5-7 h post-infusion. ATP regeneration of the infused cells was rapid with a mean increase of 1.2 mumol/g Hb above post-storage levels being achieved 1 h following transfusion.     

Interpretation of red cell survival data for in vivo compatibility testing: a normal value study

Short-term survival studies are sometimes required to determine the compatibility of donor red cells. The results of these studies are generally expressed as per cent survival at 60 min. The present study was undertaken to investigate the potential for more sophisticated data analytical techniques to improve sensitivity. In one group of eight healthy male volunteers, autologous red cells were labelled with 51chromium and injected immediately, while in a second group, red cells were stored for 5 d prior to injection. In both groups, eight to 10 samples were collected in the first 6 h and another 10-12 samples over the next 4 weeks. Estimation of the 60 min per cent survival was insufficiently sensitive to detect 'physiological' haemolysis following injection of 5-d-old autologous blood. Regression analysis of 6 h survival data, however, demonstrated significantly higher red cell clearance rates in these cases than in those receiving fresh cells, with a mean 24 h loss of 3.3% of activity. The upper limit for the 6 h red cell clearance rate was 1.63%/h after fresh autologous blood and 2.43%/h after 5-d-old blood. The significance of these findings is discussed and a protocol suggested for the analysis of short-term red cell survival data.     

The Application of Freeze-Cleaving Technics to Studies on Red Blood Cell Fine Structure

A simplified freeze-cleave and replication method of tissue preparation forexamination in the electron microscope is applied to studies on red blood cellfine structure. With this technic, the cytoplasm of red blood cells appearsto have a uniform pattern of packed granularity, with individual particles approximating the dimensions anticipated for replicas of individual hemoglobinmolecules. The cell surface is smooth and partially covered with small particles which may represent antigens, enzymes, or some structural proteins.The possibility that particles seen in cells and on cell membranes may represent an artifact is discussed. Pretreatment of cells prior to freezing influencesthe plane of cleavage through packed cells so that the plane of cleavage canbe preferentially directed either through the cytoplasm or along red cellmembranes. The freeze-cleave technic may be of particular value in applications where extensive areas of membrane must be surveyed, such as searchingfor leukemogenic viruses budding through cell membranes.

Submitted on August 16, 1966 Accepted on November 24, 1966     

The anemia of trauma

1. The anemia of civilian trauma (mainly limb injuries) has been studied in57 patients.

2. In 22 patients the red cell volume, after primary blood loss has probablyceased, has been compared with the follow-up "normal" red cell volume. Thered cell volume credit or deficit is taken as an index of the balance betweenprimary blood loss and blood transfused, and was found to be the major factorcontributing to the later polycythemia or anemia. The evidence suggests that ananemia of trauma due to unreplaced primary blood loss is still a common findingin civilian injuries today.

3. A further red cell volume estimate in 20 patients on the 4-14th day afterinjury showed on average a further fall of 11 per cent of the red cell volume.Evidence is presented for considering that there is a further disappearance ofred cells after primary blood loss has ceased in all patients—even in those whosered cell volume did not fall.

4. The evidence suggests that the anemia of trauma is largely preventable byadequate blood transfusion in the majority of civilian injuries.

Submitted on August 15, 1955 Accepted on October 15, 1955     

Separation and differential enzymic inactivation of the F and V isoantigens of the bovine erythrocyte

Summary. A subunit of the bovine erythrocyte membrane which resulted from ether and alkali extraction of stroma was found to retain possibly all of the blood group factors of the intact red blood cells. Factors in the A, B, C, F, L, S and Z systems were detected. Papain degradation of this elinin preparation resulted in loss of all detectable factors except F, V and C when they were present in the source material. The F and V properties were clearly separated from each other. The F property remained with the insoluble residue while the V was found in soluble form in the reaction supernatant. The V factor could also be released from stroma by similar papain treatment. The F factor was destroyed by treatment with neuraminidase while no other factor was found to be affected. There was evidence that more sialic acid could be released by neuraminidase or by acid hydrolysis from F-positive than from F-negative erythrocyte stroma.