Plasma adenine and cellular ATP in red cell concentrates collected and stored in modified CPD at 4 C

Eight units of blood were drawn into modified CPD containing 25 per cent higher glucose and 17.3 mg adenine (0.25 mM in blood). Red blood cell concentrates (RCC) were prepared to a mean hematocrit (Hct) of 70, the cells stored at 4 C, and plasma adenine and red blood cell adenosine triphosphate (ATP) were measured weekly for 42 days. The removal of plasma in the preparation of RCC reduced by 39 per cent the available adenine. As a result measurable plasma adenine was depleted by 21 days. The loss of ATP in RCC occurs at a significantly faster rate than in whole blood stored under the same conditions. When red blood cells are stored at higher HCT or for periods longer than 35 days, increased anticoagulant adenine levels are recommended.     

A Clinical Evaluation of the Use of Citrate-Phosphate-Dextrose Solution in Children

Red cells collected in CPD anticoagulant have been shown to have a mean postinfusion survival of greater than 75 per cent after 28 days of storage at 4C, in contrast to blood collected in ACD solution whose mean survival is less than 70 per cent after 28 days.
In a clinical study, 3,704 units of CPD blood collected in double plastic blood packs were utilized for 4,354 transfusions in 2,425 children. Evaluation of the stability of red cell antigens disclosed that blood preserved in CPD showed a minimal loss of antigenicity after 28 days. In contrast, clotted samples were hemolyzed and demonstrated marked loss of antigenicity for Kell, hr'(c) and P after 14 days.
Whole blood preserved in CPD provides two basic advantages over the presently used ACD solution. It provides a more physiologic blood for the recipient and has decreased the outdating percentage by better than 65 per cent.     

The in vitro evaluation of modifications in CPD-adenine anticoagulated- preserved blood at various hematocrits

Erythrocytes stored in the new CPD-adenine anticoagulant (CPDA-1) barely met the 70 per cent 24-hour postinfusion 51Cr recoveries on day 35 when stored at hematocrit greater than or equal to 75 per cent. CPDA- 1 differs from CPD in that it has 1.25 times the glucose concentration plus 17.3 mg adenine/63 ml. In an effort to improve the survivability (or viability) of red blood cells following extended storage (35+ days), two new CPD-adenine anticoagulants have been tested in vitro. CPDA-2 and CPDA-3 (both of which contain 34.6 mg/63 ml of anticoagulant or 0.50 mM adenine [final blood concentration], and either 1.75 times or 2.0 times respectively the amount of glucose used in CPD) have been tested for whole blood or red blood cell storage to 42 days. Red blood cell ATP concentrations were better maintained throughout 42 days of storage in both of these formulations than in CPDA-1 at hematocrits that ranged from 40 to 85. Other biochemical parameters (2,3-DPG, pH, plasma hemoglobin) were similar to those of blood stored in CPD or CPDA- 1.     

Adenine in Blood Preservation: Posttransfusion Viability and Biochemical Changes

Posttransfusion viability was studied in red blood cells stored for 21 days in ACD solution and for 35 days in ACD solution supplemented with adenine to a final concentration of 0.5 mM. The survival of radio-chromium-labeled red cells was determined after transfusion of 10 ml. of autologous blood and 350–400 ml. homologous blood. The viability values were about the same for the two transfusion procedures. The mean posttransfusion viability was 80 per cent for erythrocytes stored for 35 days in the medium containing adenine and 79 per cent for cells preserved in ACD solution for 21 days.
The concentration in the erythrocytes of ATP, ADP, AMP, reduced and oxidized glutathione, 2,3-diphosphoglycerate, and four glycolytic enzymes was measured before and after storage in the two media. The ATP and the total adenine nucleotide concentrations were much higher in the red cells stored in the adenine-containing solution. Of the enzymes tested, only phosphofructokinase decreased in activity during 35 days of storage. The decrease was about 50 per cent and was not dependent on the storage solution.
This study supports the theory that decreased adenine supply is an important cause of damage to erythrocytes in ACD solution.     

Viability and function of platelet concentrates stored in CPD-adenine (CPDA-1)

The effective use of CPDA-1 as an anticoagulant in routine blood banking practice requires demonstration that platelet concentrates prepared in this solution meet both in vitro quality control standards and maintain posttransfusion viability and function after storage. In this study of 138 units of CPDA-1 platelet concentrates, the average platelet count was 8.0 +/− 0.2 × 10(10) with 81 per cent of the units having greater than 5.5 × 10(10) platelets. The mean poststorage pH was 6.68 +/− 0.03 and only four of the units had a pH of less than 6.0 (3%). Residual plasma volume averaged 75 +/− 1 ml. Platelet viability was determined in 16 normal volunteers by measuring survival of 51Cr- labeled autologous platelets after storage for 72 hours at 22 +/− 2 C. Platelet recovery averaged 50 +/− 4 per cent, while survival was 7.3 +/− 0.4 days for the 15 units with a pH above 6.0. Measurements of posttransfusion platelet viability and function were made in 12 paients with thrombocytopenia secondary to marrow failure. Their mean pretransfusion platelet count was 17,000 +/− 2,000/microliter, and their standardized template bleeding times were all greater than 30 minutes. Platelet recovery averaged 44 +/− 5 per cent and survival 3.3 +/− 0.5 days. In seven of the patients with the best posttransfusion increments, bleeding time was improved. Five patients with poor posttranfusion platelet increments showed no improvement in bleeding time with CPDA-1; two of these patients were also transfused with CPD platelets and had no response. Our studies indicate that platelet concentrates prepared in CPDA-1 meet in vitro quality control standards and after transfusion, maintain viability and function comparable to that of CPD collected platelets.     

Di-2-ethylhexyl phthalate (DEHP) and mono-2-ethylexyl phthalate (MEHP) accumulation in whole blood and red cell concentrates

Plasma DEHP concentrations were measured weekly in whole blood and red cell concentrates (RCC) during 21 days of storage in standard CPD within PL-130 blood bags. In addition, DEHP and MEHP accumulation patterns were investigated in blood stored for 42 days in modified CPD with adenine within PL-146 and BB-69 storage containers. Total per-unit plasma DEHP of RCC units was 49 to 71 per cent of the total in plasma of whole blood units (PL-130). From 28 to 42 days, mean DEHP levels were 12 to 19 per cent higher in whole blood stored in PL-146 than in BB-69. Although MEHP was not found in any blood bag plastic, MEHP accumulated in plasma during whole blood storage. MEHP concentrations were 2.8 to 3.8 times higher in plasma stored in BB-69 than in PL-146. It is postulated that MEHP arises from hydrolysis of DEHP by plasma lipase, even in frozen plasma sample, and that the rate of this reaction is influenced by blood bag plastic surface characteristics.     

Accumulation of DI-2-Ethylhexyl Phthalate (DEHP) in Whole Blood, Platelet Concentrates, and Platelet-Poor Plasma

The concentration of the plasticizer, di-2-ethylhexyl phthalate (DEHP), in the plasma was measured after storage of the whole blood in polyvinylchloride plastic bags at 4 C for up to 38 days in either ACD or CPD. The plasma from ACD-stored whole blood contained more DEHP than that from CPD-stored whole blood. The continuous-flow centrifugation washing procedures removed about 98 per cent of the DEHP from ACD whole blood stored for 33 days at 4 C.
DEHP was assayed in the platelet-rich plasma, platelet-poor plasma, supernatant of the platelet concentrate, washed platelets, and washed red blood cells prepared from CPD whole blood. Very little DEHP was found in the washed red blood cells and platelets, a small amount was found in the platelet-poor plasma, and a large amount in the supernatant of the platelet concentrate. A greater amount of DEHP accumulated in the platelet concentrates that were stored at 22 C than in those stored at 4 C. When platelet concentrates from CPD whole blood were stored at 22 C for 72 hours, the amount of DEHP was about four times that observed after 4 C storage for the same length of time.     

Comparative Viability of Blood Stored in ACD and CPD

The normal distribution of red blood cell viability for ACD and CPD units stored for 21 days was studied. For ACD and CPD units, respectively (n=41,37), the means and standard deviations were as follows: 24-hour survival, 75.7 + 6.2 and 79.4 + 6.4; early recovery, 91.6 + 3.6 and 94.1 + 3.5; t/2, 29.4 + 3.0 and 27.9 + 4.3. Early recovery and survival were significantly higher for CPD, but more important than the difference in mean survival is that by current standards of acceptability, the incidence of donors who will be deemed undesirable is approximately 6 per cent for CPD, as opposed to 20 per cent for ACD. Neither early recovery, 24-hour survival, nor t/2 could be shown to correlate with pH, plasma potassium, plasma sodium, per cent hemolysis, and osmotic fragility. The mean and standard deviation of survival for 18 units of 28-day-old CPD blood was 70.7 + 11. Since the standard error was large, the frequency distribution could not be determined, and the number of units with survivals that would fall below the minimum standard could not be ascertained. Nevertheless, comparison with 21-day old ACD did not show a significant difference in the mean survival, although the range observed was much wider. The results also point out the need for greater number of observations with increasing duration of storage for adequate appraisal of blood preservative solutions.     

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.     

Effects of Adenine on Clotting Factors in Fresh Blood, Stored Blood, and Stored Fresh Frozen Plasma

The addition of small quantities of adenine to whole blood may prolong the useful shelf life of bank blood. The present study was undertaken to evaluate the effects of adenine on the clotting factors in blood containing ACD and CPD. Units of whole blood were collected in ACD, ACD-adenine, CPD and CPD-adenine, and each was stored 42 days under standard blood bank conditions. Samples of fresh frozen plasma containing these anticoagulants were stored three to four months at -30 C. Assays of Factors V, VIII (AHF), IX (PTC), X (Stuart Factor), fibrinogen, and prothrombin were performed on fresh blood, stored blood and stored fresh frozen plasma. The presence of small quantities of adenine did not appear to produce any appreciable alteration in the activity of the clotting factors in fresh blood. Further, adenine did not appear either to improve or worsen the survival of the procoagulants in whole blood stored 42 days or in fresh frozen plasma stored three to four months. There was significant deterioration of Factors V and VIII in whole blood stored 42 days in ACD, ACD-adenine, CPD, and CPD-adenine, but the degree of storage loss was independent of the anticoagulant employed. Factor X, fibrinogen, and prothrombin remained stable in blood stored 42 days regardless of the anticoagulant used, but Factor IX activity increased during storage possibly as the result of contact activation. Fresh frozen plasma stored three to four months showed a uniform slight loss of Factor VIII in all four anticoagulants, but Factors V, IX, X, fibrinogen, and prothrombin remained stable in stored fresh frozen plasma regardless of the anticoagulant employed.     

Red blood cell preservation in protein-poor media. I. Leukocyte enzymes as a cause of hemolysis

Red blood cells were prepared from CPD whole blood concentrated to 90 per cent hematocrit, diluted with saline-adenine-glucose (SAG) media and then stored for 35 days. The dilution was undertaken to improve the flow properties of the blood and to provide the cells with glucose and adenine allowing prolonged storage. Several different compositions were tested. ATP could be maintained at about 70 per cent of the initial level and the 24 hour red blood cell posttransfusion survival was 82 per cent. The leakage of potassium was less than in CPD-adenine whole blood if the dilution volume was half or less of the red blood cell volume. The only problem was that the hemolysis was greater in SAG-stored blood than in CPD whole blood or undiluted CPD red blood cell concentrate. Hemolysis could be reduced by removal of huffy coat cells or by storage of blood in the presence of synthetic enzyme inhibitors. A chymotrypsin-like enzyme isolated from human leukocytes had a potent hemolytic effect. The hypothesis is presented that red blood cells stored in an electrolyte medium in the presence of leukocytes undergo increased hemolysis because they lack the protecting effect of plasma enzyme inhibitors which normally inhibit any hemolytic enzymes leaking from damaged leukocytes. The study has practical implications for the prolonged storage of buffy-poor red blood cell preparations to be used in transfusion therapy.     

Therapeutic effectiveness and safety of outdated human red blood cells rejuvenated to restore oxygen transport function to normal, frozen for 3 to 4 years at −80 C, washed, and stored at 4 C for 24 hours prior to rapid infusion

Human red blood cell concentrates with hematocrit values of 75 V% were prepared from citrate-phosphate-dextrose (CPD) blood, stored at 4 C for 20 to 28 days, and biochemically modified with a solution containing pyruvate, inosine, glucose, phosphate, and adenine (PIGPA Solution A). The rejuvenated red blood cells were frozen with 40% W/V glycerol in a polyolefin plastic bag and were stored at ?80 C. After three to four years of frozen storage, the units were thawed, washed, and stored at 4 C in a sodium chloride-glucose-phosphate solution for 24 hours prior to transfusion. Red blood cell recovery was 97 per cent after thawing and 90 per cent after washing. An automated differential agglutination procedure (ADA) showed 24-hour survival values of about 80 per cent, and long-term survival values of about 85 days depending on the disease state of the recipient. The red blood cells had normal affinity for oxygen on the day of transfusion. Plasma hemoglobin levels measured immediately after transfusion indicated extravascular removal of nonviable donor red blood cells. There was no increase in the uric acid level during the 24-hour posttransfusion period. A pool of three to ten units of rejuvenated washed previously frozen red blood cells was transfused rapidly to each of 19 anemic elderly patients. The red blood cells which had normal oxygen delivery capacity immediately upon transfusion increased the recipient's red blood cell mass and produced no untoward effects.     

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.     

Hemoglobin Function of Blood Stored at 4 C in ACD and CPD with Adenine and Inosine

Normal hemoglobin function depends on adequate erythrocyte levels of 2,3‐diphosphoglycerate (2,3‐DPG), a compound that is poorly maintained during blood bank storage in acid‐citrate‐dextrose (ACD). Since 2,3‐DPG is better maintained at the higher pH afforded by citrate‐phosphate‐dextrose (CPD), hemoglobin function was compared during storage in CPD and ACD. Further, hemoglobin function was studied in CPD blood containing adenine and inosine, compounds that provide metabolic energy and thus prolong the shelf‐life of blood, because they also effect the levels of 2,3‐DPG during storage. Hemoglobin function, expressed as the P₅₀ (the P₀₂ at 50 per cent oxygenation, an inverse but direct measure of oxygen affinity) is considerably better maintained during storage in CPD than in ACD. The hemoglobin function or P₅₀ of blood stored in CPD‐adenine is not maintained as well as blood stored in CPD without adenine, but the oxyhemoglobin dissociation curves show only a small difference when compared to the difference between ACD and CPD. Blood stored in CPD‐adenine with inosine, present initially or added at day 25, allows higher P₅₀ values late in storage, thus providing better hemoglobin function for more of the storage period.     

Specific Agglutinability of Erythrocytes from Whole Blood Stored at 4 C

When whole blood is supplemented with adenine, the erythrocytes are suitable for transfusion after at least 35 days of storage at 4C, but information is not available as to whether such cells remain satisfactory for blood typing and pre transfusion tests for evidence of incompatibility. Accordingly, we designed experiments to evaluate by AutoAnalyzer the specific agglutinability of erythrocytes obtained from whole blood stored at 4C. Seven normal volunteer donors of both sexes, aged between 22 and 31 years, were chosen and bled periodically so that, over a three-day testing period, all blood specimens from each donor could be evaluated. Each donor provided at each bleeding sufficient blood to permit aliquots of 20 ml to be stored as clotted blood and 8 ml to be stored as citrated whole blood. Four kinds of citrate solution were used: ACD Formula A, CPD, ACD supplemented with adenine, and CPD supplemented with adenine. All clotted specimens were tested after 0, 1, 2, 3, 4, and 5 weeks of storage, while all citrated specimens were tested after 0, 1, 2, 3, 4, 5, 6, 8, and 10 weeks of storage. Six blood group systems were used for evaluation with the following reagents: anti-A or anti-B, anti-Rhl (Rh_o or D), anti-K2 (k or Cellano), anti-Jk^a, anti-Fy^a or anti-Fy^b, and anti-M. Each reagent was used at a dilution to support from 20 to 80 per cent agglutination with freshly drawn blood, and each reagent was tested under two conditions: low ionic and normal ionic. The results disclosed loss of specific agglutinability in association with the time of storage of whole blood at 4 C. The onset and degree of loss depended both on the kind of test used (normal ionic tests showed loss 2.8 weeks earlier than did low ionic tests) and the condition in which the blood was stored (loss occurred sooner and then progressed more with clotted blood than with citrated blood). Average losses of 25, 50, and 75 per cent were observed at 1.1, 2.1, and 4.0 weeks of storage for normal ionic tests of clotted blood, and at 4.1, 6.0, and 7.6 weeks for similar tests of citrated blood. Loss of specific agglutinability during storage was significantly less for the red blood cells of some donors than for others, but systematic differences between specific blood-typing tests were not observed. ACD and CPD solutions behaved similarly, while adenine supplementation exerted a slight preservative effect after 6, 8, and 10 weeks of storage. Loss of specific hemagglutinability of stored cells was not due to loss of specific antigenic sites because stored cells were as effective as fresh cells for the absorption of anti-B, anti-Rhl, and anti-M. Loss might, however, be related to an increase in the accumulation of IgG, fibrinogen, and albumin at the surfaces of erythrocytes.     

The distribution and utilization of adenine in red blood cells during 42 days of 4 C storage

The fate of adenine in CPD whole blood (17.3 mg/500 ml, or 0.25 mM) was evaluated during 42 days of 4 C storage. In whole blood, 95 per cent of the adenine was removed from the plasma by 42 days while the cellular adenosine triphosphate (ATP) levels remained above 60 per cent of the initial concentration. Packed red blood cells (concentrates) were stored with the same relative quantity of adenine (0.1 mg/ml red blood cells) used in whole blood units by the addition of adenine after packing and were shown to take up adenine in a similar manner. Calculations of the initial adenine distribution indicated higher intracellular adenine concentrations than predicted from distribution equilibrium based on volume considerations. The presence of inorganic phosphate has marginal effects on adenine incorporation but does elevate ATP levels, while contributing to the reduction of 2,3-diphosphoglycerate (2,3-DPG) content. The free adenine equilibrium between plasma and red blood cells favors the red blood cells, suggesting adenine binding by red blood cell membranes as shown by initial distribution studies with ¹⁴C-adenine and equilibrium dialysis.     

Comparison Studies of Whole Blood Stored in ACD and CPD and with Adenine

Using 70% 24-hour posttransfusion survival as one of the criteria of preservation, ACD and CPD anticoagulant solutions with and without adenine were tested after storage for 28, 35, or 42 days. At 28 days, all solutions had average survivals of 70% or better. The average survival values for the ACD and CPD solution groups were less than 70% at 35 and 42 days. However, both solutions with adenine had a group average value over 70% even after 35 or 42 days of storage. The average survival values between the two anticoagulants alone, were not significantly different at each time period. The two anticoagulants when adenine was included had increased survival and there was no statistical difference between the levels. In comparing two units obtained from the same subject, survival percentages were significantly higher in almost every recipient when the adenine-supplemented stored blood was used. Other chemical determinations did not show significant alterations and no toxic effects were observed in the recipients.
Since all units containing adenine had survival values greater than 70% in the 28- and 35-day periods, these units would appear to have been effectively preserved and blood stored under these conditions could be used in routine transfusions, reserving units stored 42 days for emergency use.     

In vivo viability of red blood cells stored in CPDA-2

The marginal viability of erythrocytes stored for 35 days as red blood cell concentrates in citrate-phosphate-dextrose-adenine-one (CPDA-1) was attributed to inadequate nutrient support with adenine and glucose. In an effort to improve the viability of red blood cells following extended storage, a new CPD-adenine and 1.4 times more glucose than CPDA-1. The efficacy of CPDA-2 was evaluated in vivo by measurement of 24-hour postinfusion recovery of 51Cr-labeled erythrocytes which had been stored as whole blood or red blood cell concentrates for 5 to 8 weeks. All red blood cell concentrates, and the whole blood units stored for 35 and 42 days, were held at room temperature for 8 hours prior to processing and/or refrigeration. CPDA-2 yielded significantly higher 51Cr survivals than CPDA-1 and exceeded the accepted criterion for anticoagulant preservative efficacy of 70 percent postinfusion survival of red blood cells after storage for a period of 42 days. Preliminary data supports possible usage to 49 days. Plasma glucose and red blood cell ATP concentration were maintained better in CPDA-2 than in CPDA-1. When compared to historical controls for CPD and CPDA-1 the data suggest that red blood cells stored in CPDA-02 will have superior viability throughout the entire storage period. CPDA-2 is a candidate to replace CPDA-1 as the anticoagulant-preservative solution of choice for red blood cell concentrates.     

Factors Influencing the 24-Hour Posttransfusion Survival and the Oxygen Transport Function of Previously Frozen Red Cells Preserved with 40 Per Cent W/V Glycerol and Frozen at —80 C

A 6.2 M glycerol solution was added directly to concentrated red blood cells before storage at — 80 C for at least two and one-half years. The glycerol was removed from the thawed red blood cells by one of four different washing procedures, and the washed cells were stored at 4 C for an additional 24 hours before transfusion. Recovery in vitro was about 90 per cent, and the posttransfusion survival was about 85 per cent. The CPD anticoagulant maintained the oxygen transport much better than ACD during storage of the red blood cells at 4 C for one week prior to freezing. Results were similar whether glycerolized red blood cells were washed in reusable stainless steel bowls, disposable polycarbonate bowls, or collapsible disposable polyvinylchloride plastic bags. The composition of the wash solution had no significant effect on the posttransfusion survival or oxygen transport function. When the washed red blood cells were stored in a sodium chloride-glucose-phosphate solution at 4 C for 24 hours before transfusion, the 24-hour posttransfusion survival and oxygen transport function was satisfactory. Freeze-preservation of red blood cells with hematocrits of about 40 V per cent and postthaw storage at 4 C for 24 hours resulted in an accumulation of supernatant hemoglobin and extracellular potassium. At the time of transfusion, the red blood cells were concentrated by centrifugation, the supernatant medium was removed, and the hematocrit adjusted to 70 V per cent.     

Studies on Stored Whole Blood II. Use of Packed Red Blood Cells

Whole blood collected in ACD or ACD with adenine was studied as units stored with or without plasma over 14 to 28 days. Comparisons were made using various tests, with posttransfusion survival in vivo being the primary guideline. The adverse effects which resulted from storage appeared more slowly in units with plasma than in units without plasma. The inclusion of adenine to either packed or nonpacked units was associated with even less evidence of storage damage. Using the criterion of over 70 per cent recovery in 24 hours as signifying adequate preservation, the average survival value for each group was better when the plasma was present, and even higher when adenine had been added. All tested groups had average values exceeding 70 per cent recovery after 21 days of storage. Blood stored for 28 days with adenine had over 70 per cent recovery with or without plasma. Though the storage damage became more apparent in units without plasma, the average survival value would suggest that the packed units were still useful after 21 days of storage, with extension to 28 days when ACD with adenine was used.