Factor VIII preservation following collection into commercial plasmapheresis systems

The two-stage method of Pool and Robinson was used in determining plasma Factor VIII activity following contact with four commercially available plastic blood and pooling bag films. Employing a two-arm double plasmapheresis technique, it was shown that Factor VIII activity in whole blood initially drawn into BB-69 and PL-130 blood bags decreased only slightly from in vivo levels for activity in PL-130 blood bags (t equal 3.2317, p. less than 0.01). Factor VIII activity fell to 98 and 90 per cent preservation levels from an initial 104 per cent in vivo activity, for BB-69 and PL-130 respectively. Factor VIII activity in plasma collected in Ellay and PL-146 pooling bags, both decreased significantly to 85 and 86 per cent, respectively, after three hours storage at room temperature (24 to 25.5 C). Following five weeks storage (-30 C) in Ellay and PL-146 pooling bags, Factor VIII activity significantly (p less than 0.005) fell to 79 and 77 per cent, respectively. No significant differences were observed when Factor VIII activity levels were compared in BB-69 versus PL-130 or Ellay versus PL-146 plastic film systems.     

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.     

The in vivo survival of red blood cells stored in modified CPD with adenine: report of a multi-institutional cooperative effort

In order to provide data in support of licensure applications for citrate-phosphate-dextrose (CPD) supplemented with adenine, a multi- institutional cooperative effort was organized to determine survivability of red blood cells subjected to prolonged liquid storage. Two manufacturers supplied plastic multiple bag blood storage containers prefilled with modified CPD (glucose 25/ greater than the normal concentration) supplemented with adenine (17.0 to 17.3 mg per 63 ml of anticoagulant; 0.25 millimolar approximate final concentration when diluted with 450 ml of whole blood for 35 days showed a mean survival of 80.53 +/- 6.44 per cent (1 SD). Both red blood cell and supernatant plasma biochemical characteristics were comparable to those reported for whole blood stored for 21 days in either acid-citrate- dextrose (ACD) or CPD. Red blood cells from 19 units stored as concentrates for 35 days (Hct 75.03 +/− 3.74%) had a mean survival of 71.38 +/− 10.3 per cent with considerable interdonor variation in survival and interlaboratory variation in some biochemical characteristics. Red blood cells from eight units stored as concentrates (Hct 75.38 +/− 4.30%) for 28 days showed a mean survival of 83.97 +/− 6.10 per cent and biochemical characteristics comparable to those reported for red blood cell concentrates stored in CPD or ACD for 21 days. Modified CPD with adenine as formulated offers an improved anticoagulant for blood banking by extending the permissible red blood cell storage period.     

Effect of heat on the conversion of di-2-ethylhexyl phthalate to mono-2- ethylhexyl phthalate in human plasma

Plasma di-2-ethylhexyl phthalate (DEHP), which accumulates during blood storage in plastic bags, gives rise to plasma mono-2-ethylhexyl phthalate (MEHP). This also in plasma samples awaiting analysis, which are no longer stored in plastic bags. Heating plasma samples at 60 C for 25 to 30 minutes in a water bath effectively halts conversion of DEHP to MEHP during subsequent room temperature storage for at least 100 hours. This result is consistent with the view that MEHP accumulation in plasma is due to enzymatic hydrolysis of DEHP by esterase (s) which can be heat-inactivated.     

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.     

The accumulation of mono-2-ethylhexylphthalate (MEHP) during storage of whole blood and plasma

The accumulation of the plasticizer di-2-ethylhexylphthalate (DEHP) in blood and blood components has been of considerable concern for some time. We have followed the accumulation of DEHP and one of its major metabolities, mono-2-ethylhexylphthalate (MEHP) during storage of whole blood, platelet-rich plasma, platelet concentrates, and platelet-poor plasma for periods ranging from 72 hours to four weeks. Both phthalates showed a progressive increase in concentration with time. While the levels of DEHP were much greater than those of MEHP, there was nonetheless a significant and continual increase in MEHP in all preparations. The highest concentrations of both DEHP and MEHP were found in the platelet-poor plasma, indicating that platelets do not have a major role in the accumulation of the phthalates in blood. The accumulation of MEHP was shown to be a direct result of the metabolism of DEHP by plasma protein(s) rather than leaching from the blood bag.     

Exposure of newborn infants to plasticizers. Plasma levels of di-(2- ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate during exchange transfusion

The exposure of newborn infants to the plasticizer di-(2-ethylhexyl) phthalate (DEHP) and its primary metabolite mono-(2-ethylhexyl) phthalate (MEHP) was studied during exchange transfusions by measuring their contents in the infused blood. Plasma concentrations of DEHP and MEHP in the blood withdrawn from the infants during the transfusions also were determined. The amounts of DEHP and MEHP inadvertently infused varied from 1.7 to 4.2 and 0.2 to 0.7 mg per kg body weight, respectively. Immediately after the transfusions, the plasma levels of DEHP in the individual infants varied between 3.4 and 11.1 micrograms per ml. MEHP in the corresponding samples ranged from 2.4 to 15.1 micrograms per ml. Judging from plasma concentrations of DEHP and MEHP during and after transfusion, there was no gradual accumulation of these substances in the plasma during the course of the transfusion. In the two infants who underwent a second exchange transfusion, significant levels of phthalates were found at 16 and 23 hours, respectively, after the first transfusion. Plasma concentrations of DEHP in these infants declined at a faster rate than those of MEHP, thus pointing to the importance of examining the pharmacokinetics of this potentially toxic metabolite.     

Effect of Transportation on the Posttransfusion Survival of Blood Stored in CPD

Studies on the effects of land and air transportation on CPD blood stored for 21 and 28 days are reported. Plasma potassium, supernatant hemoglobin, per cent hemolysis, whole blood pH, plasma sugar, posttransfusion survival (autologous) and t/2 of the transported units did not differ significantly from those of the control units that were left undisturbed in the refrigerator for comparable duration of storage. No untoward reactions were encountered in any of the subjects.
In addition, two subjects showed satisfactory red cell survival (above 70%) after 21 days of storage, in spite of collection deficits of 200 and 130 ml. No correlation could be shown between survival and t/2 and such in vitro parameters as per cent hemolysis, plasma hemoglobin, plasma sugar, pH and plasma potassium. A good positive correlation was found between survival and initial slope.     

Dispositions of di- and mono-(2-ethylhexyl) phthalate in newborn infants subjected to exchange transfusions

The dispositions of the plasticizer di-(2-ethylhexyl) phthalate (DEHP) and its primary metabolite mono-(2-ethylhexyl) phthalate (MEHP) were studied in newborn infants subjected to exchange transfusions. During a single exchange transfusion the amounts of DEHP and MEHP infused ranged from 0.8-3.3 and 0.05-0.20 mg kg-1 body weight, respectively. There were indications that about 30% of the infused DEHP originated from parts of the transfusion set other than the blood bag. Approximately 30% of the infused amount of DEHP was withdrawn during the course of each transfusion. Immediately after the transfusions the plasma levels of DEHP ranged between 5.8 and 19.6 micrograms ml-1, and subsequently they declined rapidly. This decline, probably reflecting distribution of DEHP within the body, was followed by a slower elimination phase. The half-life of this phase was approximately 10 h. The maximal plasma levels of MEHP were about 5 micrograms ml-1. In one pre-term infant the elimination of MEHP was slower than its formation, whereas in one full-term newborn the formation appeared to be rate-limiting for the elimination.     

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.     

Platelet storage. Effects of leachable materials on morphology and function

A polyolefin plastic (PL 732) bag formulated without liquid plasticizer allows storage of platelets for 5 and, now, up to 7 days. In order to assess the leaching of compounds from this new plastic, extracts of the supernatant from platelet concentrates stored in these bags were analyzed by high-performance liquid chromatography, mass spectrometry, and gas-liquid chromatography. A leachable material was detected and identified as di(2-ethylhexyl) phthalate (DEHP). During the sterilization process, migration of the DEHP occurs from the polyvinylchloride (PVC) bags into the PL 732 plastic bag. The level of DEHP was 12-fold less in the extracts of PC supernatant stored in the PL 732 bag than those in the polyvinyl chloride (PL 146) plastic bags which were used previously for platelet storage. Platelets stored in low DEHP concentrations in the PL 732 bags were composed of 10 to 35 percent of unclassifiable shapes. These shape changes were not observed in higher concentrations of plasticizer, although the morphology scores decreased during storage in PL 146 as well. This effect on morphology was not related directly to the dose of DEHP. When platelet membranes were isolated from platelets stored in the presence of radiolabeled DEHP, the amount of bound 14C-DEHP was found to be directly proportional to the concentration of DEHP in the plasma supernatant. However, while there was a linear relationship between the protein concentration in the membrane fraction and the amount of bound DEHP, no specific DEHP binding site could be identified by electrophoresis of the solubilized platelet membranes.     

Manufacture of red cells in additive solution from whole blood refrigerated for 5 days or remanufactured from red cells stored in plasma

Background and Objectives: To investigate methods for the production of red cell concentrates (RCC) in saline, adenine, glucose and mannitol (SAG‐M), from whole blood or red cells stored in plasma for 5 or 6 days and to provide evidence that exchange transfusion RCC in citrate phosphate dextrose (CPD) plasma or citrate, phosphate, dextrose, adenine (CPDA‐1) plasma are of comparable quality. Methods and Materials: Ten RCC in SAG‐M were produced following the remanufacture of red cells in CPD plasma on day 5/6 or after 5 days hold as leucodepleted CPD whole blood. In addition, 10 RCC in CPD plasma and 9 in CPDA‐1 plasma were stored without further processing. Units were assessed for red cell parameters including haemolysis, adenosine triphosphate (ATP), 2,3‐diphosphoglycerate (2,3‐DPG) and extracellular potassium. Results: Units in SAG‐M produced by remanufacture of RCC in plasma or by delayed manufacture of whole blood had comparable levels of haemolysis, ATP and 2,3‐DPG. Furthermore, these units underwent biochemical changes similar to reference SAG‐M units, with the exception of haemolysis which was greater at the end of shelf life and supernatant potassium which was lower following remanufacture. As expected, the decline in ATP was greater in red cells stored in CPD plasma compared with CPDA‐1 plasma. In general, units in CPD plasma were of similar quality at day 28 compared to those in CPDA‐1 plasma at day 35. Conclusions: RCC produced following the remanufacture of RCC in plasma or the delayed manufacture of whole blood are of acceptable in vitro quality and should be assigned the same shelf life as standard RCC in SAG‐M.     

In vitro properties of red cells prepared from half-strength citrate CPD/RAS-2 (Erythro-sol) donations in PL-146 plastic

Whole blood donations were collected into 0.5CPD anticoagulant in PL-146 plastic. This was shown to improve the stability of plasma FVIII levels when compared with CPD. RAS-2 was used as additive and this improved the in vitro properties of the red cells, such that post processing 2,3-DPG levels were maintained for 21 days and ATP levels were maintained for 28 days. Whether or not such improvements in red cell properties yield a benefit in clinical use remains to be established.     

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.     

Citrate-Phosphate-Dextrose Solution for Preservation of Human Blood: A Further Report

A comparison of clinically significant in vitro characteristics of ACD and CPD blood is presented.
In clinical trials conducted at three hospitals, 586 units of blood collected in CPD in plastic bags were utilized for 622 individual treatments as single units or subdivided aliquots of whole blood, and as packed red cells in 332 patients. The expiration period was 28 days, and five bloods were discarded as outdated. There was one reported pyrexial reaction and one proven bacterial contamination.
Forty-two per cent of the bloods or components were issued during the first week, 43 per cent during the second and third weeks, and 15 per cent during the fourth week of storage.
Of the total bloods or components issued, 9 per cent of the whole bloods, 25 per cent of the subdivided blood aliquots, and 23 per cent of the packed red cells were transfused during the fourth week of storage.
No difficulty with clotting was observed during collection, storage, or transfusion.     

Blood preservation XXVI, CPD-adenine packed cells: benefits of increasing the glucose

In searching for the optimal glucose concentration, this lab has monitored ATP, 2,3-DPG, pH, and glucose levels of samples taken from full blood units stored for 6 weeks at 4 C. The blood was collected into CPD-adenine containing 100, 125, 150, 175, and 200 per cent of the glucose present in CPD. The units were stored as whole blood, soft packed (50 to 70% Hct), or hard packed units (80 to 95% Hct). ATP values in general did not decrease very greatly in whole blood units and only moderately in soft packed units. However, in hard packed units a steady progressive decrease in the ATP values was seen to begin at day 14. In these hard-packed units the only improvement with extra glucose was seen beginning at day 14 when ATP maintenance was better with 200 per cent glucose, but the improvement was not significant until day 42. However, at 35 days the ATP values for 200 and 175 per cent glucose were noticeably better than for the other preservatives. Therefore, it appears from this study that the glucose concentration in CPD-adenine for hard-packed cells should be at least 175 per cent of that in regularly formulated CPD. Also, there would appear to be an advantage of having 200 per cent glucose in those units of blood that may be stored beyond 35 days for emergency blood shortage times.     

Fate in humans of the plasticizer, di-2-ethylhexyl phthalate, arising from transfusion of platelets stored in vinyl plastic bags

Platelet concentrates were shown to contain 18 to 38 mg/dl of a phthalate plasticizer (DEHP) which arose by migration from the vinyl plastic packs in which the platelets were prepared and stored. Transfusion of these platelets into six adult patients with leukemia resulted in peak blood plasma levels of DEHP ranging from 0.34 to 0.83 mg/dl (approximately 0.02 mg/dl plasma per mg DEHP administered per square meter of surface area). The blood levels fell monoexponentially with a mean rate of 2.83 per cent per minute and a half-life of 28.0 minutes. Urine was assayed by a method that would measure unchanged DEHP as well as all phthalic acid-containing metabolites. In two patients, at most 60 and 90 per cent of the infused dose, respectively, was excreted in the urine collected for 24 hours posttransfusion. These estimates, however, could be high due to the simultaneous excretion of DEHP remaining from previous transfusions or arising from uncontrolled environmental exposures.     

In vitro function and in vivo viability of stored platelet concentrates. Effect of a secondary plasticizer component of PVC storage bags

Previous studies have shown that CL-2399 (Cutter) and PL-130 (Fenwal) polyvinyl chloride (PVC) plastic bags are unsatisfactory for storage of platelet concentrates (PC) at 22 degrees C. In an effort to explain the effects of plastic bags, the chemical make-up of CL-2399 and PL-130 PVC films was determined and compared with that of P1-146 (Fenwal) PVC, which is satisfactory for PC storage at 22 degrees C. The only significant difference between the three materials was the incorporation of tetrahydrofurfuryl oleate (THFO) as a secondary plasticizer in CL-2399 and PL-130. The response of platelets to aggregating agents, uptake of serotonin, recovery from hyptonic stress, and serotonin release during storage following storage in a modified CL- 2399 plastic prepared without THFO and designated CL-3000 (Cutter) was equivalent to PL-146 and far superior to CL-2399. In vivo studies in two laboratories of platelets stored in CL-3000 bags showed satisfactory recovery (56 +/− 4.2% and 46.7 +/− 2.7%) and survival (6.4 +/− 0.4 days and 7.4 +/− 0.6 days). From these studies we conclude that the THFO secondary plasticizer component of PL-130 and CL-2399 is the cause of the poor platelet viability of platelets stored in these plastic bags. The mechanism of impairment is not known. The causative agent(s) may be degradation products of THFO (formed during manufacture of the PVC film) that are leached from the plastic into PC during storage.     

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.     

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.