ACD Blood Research Articles

Interactions of cooling rate and protective additive on the survival of washed human erythrocytes frozen to −196 °C

Washed human erythrocytes were cooled at different rates to −196 °C in the presence of different concentrations of both penetrating and nonpenetrating cryoprotective additives, and thawed rapidly. The protective effect of albumin when ACD blood is frozen was demonstrated. All compounds tested (glycerol, dimethylsulfoxide, sucrose, bovine serum albumin and polyvinylpyrrolidone) protected the red cells at freezing rates less than that giving optimal survival, and with the exception of polyvinylpyrrolidone, all compounds in high concentration increased freezing damage at faster cooling rates. The net result was to shift the cooling rate giving optimal survival to slower values. The increase in damage at faster rates in high concentrations of the nonpenetrating agents sucrose and bovine serum albumin was unexpected and casts some doubt on the idea that under these conditions nonpenetrating compounds protect the cells by withdrawing water that could otherwise form intracellular ice.

The post-thaw viability of red blood cells of ACD and CPD blood preserved in the frozen state with and without added adenine.

Bloods collected in ACD and CPD were frozen by the Blood Research Institute Glycerol method using the Latham Processor. Washed, thawed red blood cells were suspended in autologous plasma with and without added adenine and stored at 4 C. Post‐transfusion survival studies by 125I, 51Cr methods had values of 70 per cent or better for seven and 14 days for ACD and CPD bloods, and 14 and 21 days for ACD‐adenine and CPD‐adenine bloods, respectively. Integration of a frozen blood program in conventional blood banks is discussed.

A new method for evalution of antithrombogenicity of materials.

AbstractA new method was developed for evaluating kinetically the antithrombogenicity of materials. The amount of thrombus formed was measured gravimetrically at an appropriate interval of time after calcium chloride was added to ACD blood in contact with a test material. Graphic curves for thrombus formation on glass, Silastic, Hydron, and treated natural tissues were obtained and compared. There was good correlation between our in vivo tests and in vivo observations.

SERUM IONIZED CALCIUM CHANGES FOLLOWING CITRATED BLOOD TRANSFUSION IN ANAESTHETIZED MAN

SUMMARY We measured the serum ionized calcium concentration in six patients who received ACD blood infusions during general anaesthesia and found the average decrease to be 0.5 mg/100 ml after 500 ml of blood and 0.6 mg/100 ml after 1000 ml of blood. However, the ion concentration increased a mean of 0.3 mg/100 ml 10 minutes following complete infusion of the blood. We also studied the effect of PaCO2 on ionized calcium level in six patients under general anaesthesia who did not receive blood intra-operatively and found that the ion concentration varied directly with the PaCO2 An equation for a regression line relating PaCO2 and calcium ion concentration was derived.

Plateletpheresis in a Disposable System

A new plastic disposable system is described for the removal of platelets from single packs of ACD blood, single units of blood directly from a donor, or multiple cycles of whole blood (plateletpheresis) from the same donor in a continuous flow system. Data are given on donor acceptability, platelet yields, and the effectiveness in vitro and in vivo of platelet suspensions so derived.

The Role of the Laboratory in Screening Patients for Open Heart Surgery

Patients scheduled for open heart surgery should have a tube of clotted blood submitted as early as possible to the blood bank laboratory for ABO, RH phenotype, cell typ ing and irregular ant ibody detection and identif ication. Thus, depending on the type of patient surgery and extracorporeal pump needs or size, appropriate units of ACD blood less than four days old can be collected and readily available the day of the scheduled operat ion.

Transfusion of long stored whole blood or washed red blood cells incubated with adenine and inosine.

Full unit autotransfusions of long stored ACD blood, incubated with adenine and inosine at 37 C., were given to healthy young male volunteers. Red cells of blood stored for 35 days showed, after regeneration, a significant increase in ATP and a 24‐hour posttransfusion survival of 78.8 per cent (70.9–85.9%); red cells of blood stored for 42 days, thus regenerated, showed a similar increase in ATP and a 24‐hour posttransfusion survival of 75.6 per cent (71.5–80.6%). These results were not significantly different from those obtained with 10‐ml token autotransfusions of blood similarly treated, the posttransfusion survival of red cells in token transfusions being 78.8 per cent for blood stored 35 days prior to regeneration with adenineinosine and 74 per cent for blood stored for 42 days prior to regeneration. Available data on toxicity of adenine and inosine have been critically reviewed: Chance of direct toxic effects with the small amounts involved may be dismissed when few transfusions are involved; however, uric acid overload must be considered when multiple transfusions are required within a short period of time. A single washing with saline‐glucose solution reduces by 90 per cent the concentration of un‐metabolized adenine and inosine, and of the product of their metabolism, hypoxanthine. The washing procedure involves a loss of only 0.55 per cent of the total red blood cell population; washing additionally reduces the amount of free hemoglobin. Washing has no effect on the ATP or red blood cell viability, and is recommended when multiple transfusions of cells treated with adenine and inosine are required in a short period of time.

Effect of exchange transfusion (ET) on serum ionized calcium (Ca++)

The citrate in ACD blood is known to complex Ca++ and calcium gluconate is usually infused during ET to counteract this effect. We have measured Ca++ with a Ca++ selective electrode during ET with non-buffered ACD and heparinized blood and have studied the effect of administering 0.1g calcium gluconate per 100 ml ACD blood exchanged. Heparinized blood produces negligible changes in Ca++, whereas with ACD blood a profound fall in Ca++ occurs even when calcium gluconate is given. Values for Ca++ during ET and ACD blood and added calcium gluconate are much lower in premature than in full term infants (p < 0.001). Omitting calcium gluconate further lowers Ca++ levels in full term infants (p < 0.05). The injection of calcium gluconate produces a transient rise in Ca++ which is rapidly neutralized by the infused citrate. Cessation of ET produces a prompt return toward normal values. No relationship was shown between Ca++, total Ca and protein. In spite of frankly hypocalcemic levels of Ca++ (particularly in prematures) no clinical evidence of tetany was seen. Ca++ levels could not be correlated with the infant's clinical state during the ET. We conclude that in full term infants the use of calcium gluconate in ET using ACD blood exerts a small but significant effect on Ca++. The clinical significance of this observation requires further evaluation.

Calcium ion activity during rapid exchange transfusion with citrated blood.

Measurement of ionized calcium levels by the technic of Soulier during massive transfusion of ACD blood supplemented with various quantities of calcium led to the apparently erroneous conclusion that ionized calcium levels would rise in the recipient when each unit was supplemented with 0.6 g CaCl2, the quantity which restores normal ionized calcium levels in vitro.Measurement of ionized calcium activity with the Orion electrode indicates that this ratio of CaCl2 will maintain an approximately normal ionized calcium level in dog recipients if it is infused simultaneously with the blood. Based on this information, it appears reasonable to assume that heparinized ACD blood, recalcified with 0.6 g CaCl2 per unit, may be used to prime the extracorporeal circuit for open heart surgery with the confidence that it will not alter the ionized calcium activity of the patient's blood.Addition of calcium to the circulation following administration of ACD blood causes a sharp rise and then a fall in the recipient's level of ionized calcium. Further studies in a variety of situations are required before final guidelines can be formulated for supplementation of ACD blood with calcium in massive transfusion.

Deduction of mean red-cell life-span from 51Cr survival curves.

Summary. Twenty‐two estimates of Cr red‐cell survival in normal subjects, using the ‘citrate‐wash’ method of labelling, are described; results obtained with this method are shown to have given closely similar results in three different laboratories. By contrast, methods in which 51Cr is added to ACD blood have given a much wider range of normal values.In 14 patients red‐cell survival was estimated simultaneously with DF32P and 51Cr, the latter results being corrected for Cr elution using empirical factors. Estimates of mean red‐cell life‐span by the two methods agreed fairly closely. It is concluded that when 51Cr is used as a red‐cell label, the citrate‐wash method, or some other method which has also been shown to give reproducible results in different laboratories, should be used and the results should then be corrected so as to obtain an estimate of mean life‐span. Such estimates should be more useful in clinical practice than uncorrected Cr survival data.

A method for the preservation of human blood group erythrocyte antigens in liquid nitrogen for a test cell panel.

Abstract. A modification of Krijnen's cryopreservation method, using low intracellular concentrations of 17.5% glycerol and extracellular 4% sorbitol as additives, has been developed for liquid nitrogen freezing of small volumes of erythrocytes for a test cell panel. A technique for their subsequent successful storage in sterile, dextrose‐electrolyte maintenance solution (CP‐2) at 4°C after recovery is presented. By these procedures, the mean recovery of red cells after lytic losses from freezing and thawing injury was 94.1%. The manipulations involved in transferring, mixing and resuspending the red cells removed an additional 11.1–16.2%, resulting in an overall recovery of 80.5% for serologic tests. Less than 3% of the cells exhibited cumulative lysis at seven days maintenance in CP‐2 solution at 4°C, and the blood group antigens examined remained as potent as those in sterile, fresh ACD blood.

Autoradiography of Diffusible Compounds in Human Nonnucleated Erythrocytes: Studies with Tritiated Glucose and Adenine

Abstract A simple method for autoradiography of diffusible substances in erythrocytes is described. It is based on retarding diffusion from the red corpuscles during fixation and processing by using the bifunctional aldehyde, glutaraldehyde; rapid drying; and applying the nuclear emulsion as a dried, preformed film. The technique was used for studying the changes during storage in the ability of red blood cells to utilize glucose. The incorporation of glucose-6-3H by samples of blood stored for different periods of time was followed by the number of silver grains present in the red corpuscles. It was observed that the percentage of erythrocytes with fewer than four grains increased progressively during the storage of ACD blood. The incorporation of glucose as observed by the silver grain pattern was reversible toward that of freshly collected red corpuscles following incubation of the stored samples with adenosine. Similar studies were performed using adenine-8-3H as the label in blood stored for six weeks in ACD adenine solution. Incubation of the blood samples containing 0.5 mM. adenine for one hour at 37°C resulted in autoradiographic patterns equivalent in grain counts to those given by red blood cells stored with the radiolabeled adenine for five weeks. The incubation of fresh samples with inosine was found to inhibit the incorporation of adenine, whereas inosine enhanced the number of grains in red blood cells which had "run down" during storage. The autoradiographic technique presented should be of value for the in vitro evaluation of methods of blood preservation and for studying the metabolic heterogeneity of normal red corpuscles and other tissue cells. It should be useful for investigating hereditary and acquired abnormalities of metabolism.

血液保存の実験的研究とくに酸素加赤血球の還元グルタチオン添加ACD液保存について

At present time, the human ACD blood preservation is ordinally permitted within 3 weeks in Japan.The purpose of this experiments are to make the more longer term preservation of blood in liquid state.In 1965, Sase reported that the denaturation of hemoglobin solution to met-hemoglobin during storage had been minimumed in oxyhemoglobin or reduced hemoglobin more than half-to-half mixture.This phenomenon might be occured in hemoglobin in Red Blood Cell during the storage.Experiment IUnder this hypothesis three kinds of ACD blood groups, first in regular venous state, second in fully oxygenated state, and third in reduced state were stored. After 2 weeks preservation in 4 to 10 centigrade room those blood were transfused in rabbits, the RBC post-transfusion survival was measured with radio-active cromium laveling method.The results of T1/2 in each oxygenated group, deoxygenated group, and venous group were 14.3 days, 6.1 days, and 11 days.Respectively, the most effective one was observed in oxygenated ACD blood group.Experiment IIExpecting the protective effect of oxydative hemolysis about RBC membrane, reduced gluthatione (GSH) (100mg/dl) was added in each three blood groups in Exp. I. After 2 weeks storage the RBC post-transfusion survival was measured.The T1/2 of the oxygenated GSH added group was prolonged to 20.3 days.The T1/2 of venous GSH added group was same to Exp. I. But the T1/2 of deoxygenated GSH added group after 3 weeks storage was prolonged to 15 days.The remarkable effect was observed in oxygenated GSH added blood group and deoxygenated GSH added blood group.Experiment IIIOxyphenarsine (0.01g/dl) which act to block the sulphohydril conpounds in blood was added in oxygenated GSH blood group.Two weeks later the blood was transfused in rabbit and RBC posttransfusion survival was measured. The T1/2 was 12 days, which resembles to that of venous blood. The results indicate the fact that SH component acts to protect the RBC membrane from denaturation. The protection was not recognized in mixed venous state.

The effect of haematocrit and anticoagulants on the rateof packing of erythrocytes by a centrifuge

The rate of packing of erythrocytes during centrifugation has been investigated over a range of haematocrits from 2 to 47%. This approximates to Stokes' Lawonly when the haematocrit is less than 8%. At 10% haematocrit significant cell-cellinteraction occurs and at normal blood haematocrits packing is brought about by theweight of cells, which are in contact, pressing down on each other. An invest'igation offour different methods of preventing coagulation suggests heparin is the least detrimen-tal during the 24 hours following collection. Defibrination with glass beads leaves thecells relatively inflexible. ACD and EDTA are similar initially and close to theheparinized cells. After 73 hours only the ACD blood is comparable with its initialrate of packing.

Diluted ACD blood for cardiopulmonary bypass in children : E. Sarkozy, A. J. Kovacs, R. L. Denton, and A. R. C. Dobell. Canad. J. Surg. 12:193–196 (April), 1969

Diluted ACD blood for cardiopulmonary bypass in children : E. Sarkozy, A. J. Kovacs, R. L. Denton, and A. R. C. Dobell. Canad. J. Surg. 12:193–196 (April), 1969

電子顕微鏡 (低倍率) による冷凍赤血球の研究

These studies were undertaken to clarify some causes of posttransfusion hemoglobinuria of frozen blood processed by modified Huggins procedure. For these purpose, the membranous characteristics of the fresh ACD, the storaged ACD and the frozen red cells were morphologically classified into four types by use of the low power (5000 ×) electronmicroscope. The criteria for the classification are basedonn theentire shapes, the thickness of the outer layers and the surface conditions with or without holes of the membranes.The following results were obtained.1. The type I is considered as the most juvenile one, and the type the most aged. The type I and II are the intermediates.2. These aging-like phenomena of the red cell membrane in vitro were occured and observed either during the preservation in 4°C as the ACD blood or during the processing of frozen blood. The type I transformes to the II the II to the III and the III to the IV. The final one is hemolysed. These aging-like phenomena might be caused by processings of freezing, thawing, washing and resuspension, however it is still in controversy which one is the most effective factor for the membrane damages. The aging-like phenomena occured remarkablly when the 14th or the later dated ACD blood after collection were used for the frozen blood.3. Considering the above results and the clinical experiences of frozen blood transfusion of 378 units for 93 patients, of whom 7 cases showed hemoglobinuria, the more juvenile red cells will be the tolerable and preferable for the processing, and the deadline of ACD blood to be used as the frozen blood showed be settled on the 7th day after collection. And, the effective volume of cryoprotective agent should, of course, be added to the red cells.4. Frozen in a glass bottle for japanese one unit transfusion which has a limited capacity of 230ml, posttransfusion hemoglobinuria might be caused from shortage of Huggins 8.6 molar glycerol solution to be added into the bottle, althought the fresh red cells in the 7th day were used.

Recent advances in preparation of platelet concentrates from ACD and CPD blood: Pert, J. H., A. Lundberg, M. B. Zuker, R. Yankee, and E. Henderson. Vox Sang., 13: 119–126, 1967. (27 ref.)

Recent advances in preparation of platelet concentrates from ACD and CPD blood: Pert, J. H., A. Lundberg, M. B. Zuker, R. Yankee, and E. Henderson. Vox Sang., 13: 119–126, 1967. (27 ref.)

Relation of shape and metabolism in human erythrocytes: I. Volume, ATP, and monovalent cation interdependence of cells incubated at 37°C

Erythrocytes of whole blood incubated at 37°C underwent large increases in average density and cellular hemoglobin (indicative of volume decrease) soon after depletion of ATP. Coincident were rapid exchanges of cell potassium with plasma sodium ions. The incubation time corresponding to loss of 90% or more of ATP correlated with the onset of cell contraction and rapid cation exchange. This time (approximately 45 hr) in whole blood preserved in ACD was diminished in heparinized blood or ACD blood containing fluoride, approximately the same in four units of previously frozen red cells, and extended by 15 or more hr in whole blood containing adenine, adenine + inosine, or adenine + inosine + guanosine.

The effect of THAM and sodium bicarbonate on the oxygen dissociation curve and pH difference across the red cell.

Extract: This report presents the difference (Δ [H+)], and ratio (Δ pH), of the hydrogen ion activity between the inside and outside of the red cell for THAM-buffered and NaHCO3-buffered ACD blood of man. Figure 2 shows that there was a significant difference in Δ pH and Δ [H+] between bloods buffered with THAM or sodium bicarbonate, this difference being due to the fact that normal Δ pH was reduced in THAM-buffered blood to a Δ pHof 0.072 (see table I). The oxygen dissociation curves were constructed at a variety of whole blood pHs for THAM-buffered and sodium bicarbonate-buffered ACD blood. As predicted from the measured difference in Δ pH, the oxygen dissociation curves were different for these two bloods. Table II presents the appropriate constants for the general equation log pO2 = k1 + k2 (7.4-pH) + k3 log (s/100−s). With the use of a computer program and Calcomp plot routine, the log (s/100−s)/log pO2 plot for THAM-buffered (fig. 3) and sodium bicarbonate-buffered ACD blood (fig. 4), together with their 95% confidence limits, were constructed. Speculation: Comparison of Δ pH measurements with oxygen dissociation curves should help in distinguishing between direct effect of acids and bases upon the oxygen affinity of hemoglobin and their indirect effect on oxygen affinity through an alteration in Δ pH across the red cell membrane. Secondly, the use of THAM to increase the oxygen affinity of whole blood may be of help to those studying fetal physiology and interested in determining whether fetal oxygen consumption is limited by an increased oxygen affinity in fetal blood.

THE EFFECT OF BUFFERED AND NON-BUFFERED ACD BLOOD ON ELECTROLYTE AND ACID-BASE HOMEOSTASIS DURING EXCHANGE TRANSFUSION

Exchange transfusions have been performed using buffered and non-buffered ACD blood. In 15 low risk infants receiving unbuffered blood, severe acidosis occurred in one during the transfusion. Other investigators have recently observed similar findings. In contrast, when blood was buffered prior to exchange transfusion in 26 high risk infants (i.e., with cardiorespiratory insufficiency), acidemia did not occur. Since the mortality of exchange transfusion is increased in infants with cardiorespiratory insufficiency, it appears that buffered ACD blood will lessen the risks. It is recommended that, ACD blood is used for exchange transfusion in any infant, the blood should be buffered immediately prior to transfusion using 10 mM of 1.0-1.2 M THAM.