Citrate Phosphate Dextrose Plasma Research Articles

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

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. 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. 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. 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 and in vivo evaluation of a whole blood platelet‐sparing leukoreduction filtration system

In-line leukoreduction (LR) filters decrease adverse clinical sequelae caused by residual white blood cells (WBCs). Such filtration, however, can remove platelets (PLTs) needed for production of PLT concentrates (PCs). This study measured in vitro and in vivo efficacy of a new whole blood PLT-sparing LR filter (WBPSF) system that performs whole blood (WB) LR using a single closed-system filtration step. The WBPSF provides three final LR products: AS-5 red blood cells (RBCs), citrate-phosphate-dextrose (CPD) PLTs, and CPD plasma. Volunteers (n=59) donated WB processed using the WBPSF system. WB filtration time was recorded, and LR WB was processed into AS-5 LR RBCs, CPD LR PLTs, and LR plasma. Final components were assayed for in vitro indices, and in vivo characteristics for LR AS-5 RBCs and CPD PLTs were assayed after radiolabeling. WB filtration time averaged 37 minutes. Transfusion products obtained after WBPSF met all in vitro and in vivo Food and Drug Administration (FDA) requirements. Radiolabeling of LR AS-5 RBCs after WBPSF showed a 24-hour RBC recovery of 81.3±5.3% after 42 days of storage. In vivo dual (111) In/(51) Cr radiolabeling of PCs manufactured using WBPSF showed a Day 5 recovery ratio of 80±19% versus fresh autologous PLTs and a survival ratio of 81±17% that of fresh autologous PLTs. All WBPSF-derived transfusion products met or exceeded in vitro and in vivo FDA guidelines. This filtration system is suitable for routine blood center or hospital use in the production of LR AS-5 RBCs, CPD PLTs, and CPD plasma.

Stability of blood coagulation factors and inhibitors in blood drawn into half-strength citrate anticoagulant.

Drawing of blood into a citrate-phosphate-dextrose (CPD) solution with a reduced citrate concentration has been shown to improve the maintenance of coagulation factor VIII (F VIII) in plasma and to give possibilities to improve erythrocyte preservation. We studied the quality of plasma obtained from whole blood drawn under continuous mixing into CPD in which the citrate concentration was reduced by 50% (0.5CPD). The blood was stored at room temperature for 8 h before component preparation. We confirmed improved stability of F VIII by 0.5CPD. We found no clinically significant changes in inhibitors to the coagulation and kallikrein systems, and no signs of activation of these systems, during the 8-hour holding time. In control blood drawn into CPD, F VIII and coagulation factor IX decreased by 0.09 IU/ml (8%) and 0.07 U/ml (7%), respectively, otherwise we found no significant differences between 0.5CPD plasma and CPD plasma.

Stability of Blood Coagulation Factors and Inhibitors in Blood Drawn into Half-Strength Citrate Anticoagulant

Drawing of blood into a citrate-phosphate-dextrose (CPD) solution with a reduced citrate concentration has been shown to improve the maintenance of coagulation factor VIII (F VIII) in plasma and to give possibilities to improve erythrocyte preservation. We studied the quality of plasma obtained from whole blood drawn under continuous mixing into CPD in which the citrate concentration was reduced by 50% (0.5CPD). The blood was stored at room temperature for 8 h before component preparation. We confirmed improved stability of F Vili by 0.5CPD. We found no clinically significant changes in inhibitors to the coagulation and kallikrein systems, and no signs of activation of these systems, during the 8-hour holding time. In control blood drawn into CPD, F Vili and coagulation factor IX decreased by 0.09 IU/ml (8%) and 0.07 U/ml (7%), respectively, otherwise we found no significant differences between 0.5CPD plasma and CPD plasma.

Direct capture of plasma factor VIII:C by ion exchange chromatography.

An ion exchange medium, DEAE-Fractogel 650M, can be used to capture factor VIII (FVIII) directly from plasma. Previous reports have focussed on the use of this medium to capture FVIII from cryoprecipitate. In this report, citrate phosphate dextrose plasma was batch-stirred with DEAE-Sephadex A50, filtered, diluted, loaded onto a column packed with DEAE-Fractogel TSK 650M, and chromatographed. Most of the unwanted proteins flowed through the gel unadsorbed. Bound FVIII was eluted by increasing the ionic strength of the buffer. A citrate-based buffer gave an overall FVIII:C yield of 670 IU/kg plasma with a specific activity of 0.68 IU FVIII:C/mg protein. This process gave a higher yield of FVIII:C but significantly more prothrombin complex factors than cryoprecipitation. This method is a promising alternative to cryoprecipitation of FVIII.

Platelet concentrates in an additive solution prepared from pooled buffy coats. In vivo studies.

Leukocyte-depleted platelet concentrates were prepared from pools of 4 buffy coats on the day after blood collection (BC-PC). The storage medium was composed of citrate phosphate dextrose plasma and a platelet-additive solution. Autologous transfusions of 111In-labelled platelets in 9 healthy volunteers were performed on the day of preparation (day 1) and on day 5. The recovery was 54.6 +/- 8.7 (day 1) and 51.9 +/- 10.4% (day 5), T1/2 was 101 +/- 28 and 61 +/- 9 h, respectively. The survival was 8.3 +/- 1.7 and 5.7 +/- 1.0 days, respectively, using linear plot, and 7.8 +/- 2.0 and 5.8 +/- 0.5 days using the multiple hit method. In a prospective clinical study a comparison of the corrected posttransfusion increments was made between BC-PCs and apheresis-PC, and between fresh (1-2 days) and stored (3-5 days) preparations. No difference was found between BC-PCs and apheresis PCs. However, fresh BC-PCs gave higher increments than stored BC-PCs. A slight numerical difference between fresh and stored apheresis-PCs was not statistically significant. It is concluded that the BC-PC method results in platelets of equal quality to apheresis-PC.

Zinc Ions Inhibit Factor I‐Mediated Release of CR1‐Bound Immune Complexes and Degradation of Cell‐Bound Complement Factors C3b and C4b

ZnCl2 exerted a dose-dependent inhibition of citrate-phosphate-dextrose (CPD) plasma-induced release of 125I-labelled BSA-anti-BSA immune complexes (IC) bound to complement receptor type 1 (CR1, CD35) in human whole blood. Maximal inhibition was observed at 10 mM of ZnCl2. Furthermore, the release of IC bound to erythrocyte (E)-CR1 by purified factor I, factor I-deficient serum plus purified factor I, or normal human serum was reduced by approximately 90%, 64%, and 52%, respectively, in the presence of 10 mM ZnCl2. The effect of ZnCl2 on factor I-mediated degradation of cell-bound C3b/C4b was also investigated employing CPD blood or E from a factor I-deficient donor. These cells expressed covalently bound C3b and C4b as demonstrated by a simple agglutination technique. Upon incubation of CPD whole blood with purified factor I, or of E with purified factor I or normal CPD plasma, the C-fragments were cleaved and the cells were no longer agglutinated by antibodies to C3c and C4c. The presence of ZnCl2 prevented this factor I-mediated degradation of C3b and C4b, as evidenced by the unaffected agglutination of the cells by the antibodies. We conclude that ZnCl2 inhibited factor I activity since: (1) release of complement-preopsonized IC from E-CR1 by purified factor I was markedly inhibited (90%) in the presence of ZnCl2, (2) preincubation of the cells with ZnCl2 caused only a moderate inhibition (32-38%) of the IC release, and (3) degradation by purified factor I of covalently cell-bound C3b and C4b was abrogated in the presence of 10 mM ZnCl2.

Human Factor VIII: A Calcium-Linked Protein Complex

The possible role of Ca2+ as an essential constituent part of the human factor VIII complex has been investigated by stability studies, metal determinations, and gel filtration experiments. In citrated plasma, the factor VIII coagulant activity (VIII:C) deteriorated during storage in a biphasic manner. Collection of blood in heparin, instead of chelating anticoagulants, or neutralization of citrate by addition of CaCl2 to heparinized citrate phosphate dextrose (CPD) plasma rendered VIII:C noticeably stable. At physiologic levels of ionized calcium, VIII:C was almost completely stable during incubation of plasma for 6 hr at 37 degrees C. The influence of other divalent ions was also studied. Highly purified factor VIII complex was subjected to atomic absorption spectrophotometric analysis and found to contain about 1.0 mole calcium per 220,000 daltons. This intrinsic calcium could be readily removed by EDTA. When heparin plasma and CPD plasma were chromatographed on Sepharose CL-6B at 37 degrees C, all the factor-VIII-related activities eluted together as large protein complexes. In contrast, factor VIII coagulant antigen (VIII:CAg) and factor-VIII-related antigen (VIIIR:Ag) were completely dissociated upon exposure to EDTA. From these observations it is concluded that human factor VIII circulates in normal plasma as a calcium-linked protein complex.

Human factor VIII: a calcium-linked protein complex

The possible role of Ca2+ as an essential constituent part of the human factor VIII complex has been investigated by stability studies, metal determinations, and gel filtration experiments. In citrated plasma, the factor VIII coagulant activity (VIII:C) deteriorated during storage in a biphasic manner. Collection of blood in heparin, instead of chelating anticoagulants, or neutralization of citrate by addition of CaCl2 to heparinized citrate phosphate dextrose (CPD) plasma rendered VIII:C noticeably stable. At physiologic levels of ionized calcium, VIII:C was almost completely stable during incubation of plasma for 6 hr at 37 degrees C. The influence of other divalent ions was also studied. Highly purified factor VIII complex was subjected to atomic absorption spectrophotometric analysis and found to contain about 1.0 mole calcium per 220,000 daltons. This intrinsic calcium could be readily removed by EDTA. When heparin plasma and CPD plasma were chromatographed on Sepharose CL-6B at 37 degrees C, all the factor-VIII-related activities eluted together as large protein complexes. In contrast, factor VIII coagulant antigen (VIII:CAg) and factor-VIII-related antigen (VIIIR:Ag) were completely dissociated upon exposure to EDTA. From these observations it is concluded that human factor VIII circulates in normal plasma as a calcium-linked protein complex.

Improved yields of factor VIII from heparinized plasma.

The Factor VIII procoagulant activity in plasma, cryoprecipitate, and their polyethylene glycol (PEG) precipitates is markedly increased if blood is collected into heparin rather than into citrate phosphate dextrose (CPD). There is a 34% increase in the initial level of the Factor VIII activity in the heparinized plasma with 78% of this initial activity (184 U) recovered in the cryoprecipitated. As well, the stability of the Factor VIII activity is improved: after 24 h of incubation at 22 degrees C, 99% of the initial activity is retained in heparinized plasma whereas only 64% remains in CPD plasma. The cryoprecipitate prepared from heparinized plasma is equally stable after 24 h. The PEG concentrate prepared from the cryoprecipitate of heparinized plasma is increased to 128 U compared to only 54 U from CPD plasma. Relative recoveries were 531 U/l for heparinized plasma versus 215 U/l for CPD plasma. This represents a 147% increase in yield.