SAG-M Solution Research Articles

Allogeneic cord blood red blood cells: assessing cord blood unit fractionation and validation.

For neonates and preterm infants, in whom a transfusion dose is low, the use of red blood cells (RBC) from cord blood appears to be feasible. Standardisation of fractionation and identification and assessment of quality control parameters for such RBC are still lacking. We describe the process used to obtain RBC from cord blood for transfusion purposes, including quality controls to evaluate fractionation performance and the effects of storage. The cord RBC, to which SAG-M was added, were sampled on the day of fractionation, and 7 and 14 days (end of storage) later in order to measure the complete blood count, biochemical parameters and residual white blood cells. We also assessed microbial contamination. Data relative to 279 cord blood units were evaluated. The median gestational age at collection was 40 weeks (interquartile range [IQR] 39.1-40.7) and the median volume was 90 mL (IQR 81-103). Units were subjected to automated fractionation with Compomat, and packed RBC were suspended in SAG-M solution. The median volume of the SAG-M-suspended units was 31 mL (IQR 24.0-38.1) and the median haematocrit was 54.2% (IQR 49.4-59.5). The median volume after leukoreduction was 22 mL (IQR 17-28), with the volume decrease being similar in units leukoreduced before (n=75) or after (n=204) storage. The haematocrit of leukoreduced units was higher than that of buffy coat-depleted units. Storage at 2-6 °C for 14 days was accompanied by an increase of potassium levels and percentage of haemolysis. Microbial cultures were positive for 2.9% of the collected units. Fractionation of whole cord blood can provide RBC concentrates with similar baseline characteristics as units from adults. The transfusion dose and quality of the units appear safe and suitable for clinical use in neonates, with a satisfactory haematocrit and residual white blood cell content, despite a very variable collection volume.

Metabolic fate of adenine in red blood cells during storage in SAGM solution.

Red blood cells (RBCs) are routinely stored and transfused worldwide. Recently, metabolomics have shown that RBCs experience a three-phase metabolic decay process during storage, resulting in the definition of three distinct metabolic phenotypes, occurring between Days 1 and 10, 11 and 17, and 18 and 46. Here we use metabolomics and stable isotope labeling analysis to study adenine metabolism in RBCs. A total of 6 units were prepared in SAGM or modified additive solutions (ASs) containing 15 N5 -adenine. Three of them were spiked with 15 N5 -adenine on Days 10, 14, and 17 during storage. Each unit was sampled 10 times spanning Day 1 to Day32. At each time point metabolic profiling was performed. We increased adenine concentration in the AS and we pulsed the adenine concentration during storage and found that in both cases the RBCs' main metabolic pathways were not affected. Our data clearly show that RBCs cannot consume adenine after 18 days of storage, even if it is still present in the storage solution. However, increased levels of adenine influenced S-adenosylmethionine metabolism. In this work, we have studied in detail the metabolic fate of adenine during RBC storage in SAGM. Adenine is one of the main substrates used by RBCs, but the metabolic shift observed during storage is not caused by an absence of adenine later in storage. The rate of adenine consumption strongly correlated with duration of storage but not with the amount of adenine present in the AS.

Storage of whole blood overnight in different blood bags preceding preparation of blood components: in vitro effects on red blood cells.

Routines for the storage of whole blood (WB) overnight for the preparation of blood components on the following day are of increasing interest primarily for logistic reasons. The present study focuses on in vitro effects during storage for 6 weeks on red blood cells (RBC) prepared in different blood containers after being held overnight. Five different blood collection systems were used with either inline leucocyte reduction red cell filters for the preparation of RBC, buffy coat (BC) and plasma or WB filters for the preparation of RBC and plasma. A new container with an integrated WB filter removing leucocytes but not platelets was also included for the preparation of leucocyte-reduced RBC, BC and plasma units. Standard CPD solution (63 or 70 mL) and SAG-M solution (100 or 110 mL) were used for the collection of either 450 or 500 mL blood. All WB units were stored at room temperature, either overnight for 18-24 hours (test groups, n=104) or for up to 8 hours (reference groups, n=20). In addition, five test units were stored overnight under refrigeration. In test groups (overnight storage at room temperature) we found significantly lower levels of extracellular potassium, 2,3-DPG and pH (up to day 28). During storage, higher levels of ATP (Terumo, CaridianBCT until day 35, Fresenius until day 14, Fenwal throughout storage) were seen in test groups than in reference groups. When WB was stored overnight at 2-6 degrees C before WB filtration, the levels of ATP and haemolysis were higher than in the corresponding reference. Significant differences in in vitro parameters were observed between RBC prepared within 8 hours and 18-24 hours after blood collection. The results were consistent irrespective of the blood container used. New alkaline solutions may decrease the differences.

A Modified Process for Preparation of Pathogen Inactivated RBC Using S-303 in Multiple Additive Solutions Maintains RBC Function and Reduces Immunologic Activity.

Background: Treatment of RBC with S-303 (acridine derivative) inactivates pathogens and leukocytes. The original process for S-303 treatment of RBC (oSRBC) for pathogen inactivation (PI) used 0.2mM S-303 and 2mM glutathione (GSH). Two randomized, controlled Phase III trials were voluntarily discontinued when antibodies, specific to residual acridine on oSRBC, were detected in 2 patients. Following this observation, a modified PI process using 0.2mM S-303 and 20mM GSH (mSRBC) was developed to reduce S-303 binding to the RBC surface which inactivates a broad spectrum of pathogens and maintains good RBC function.Aims: This study was designed to demonstrate the reduction in binding of S-303 to the RBC surface and preservation of in vitro RBC function with MSRBC in preparation for Phase I clinical trials.Methods: Leukoreduced RBC were prepared in Erythrosol, AS-3, AS-5, SAG-M or PAGGS-M additive solution. mSRBC were treated with 20mM GSH, pH 9.0, plus 0.2mM S-303 and incubated for 20 hours at RT. Post-treatment binding of S-303 to the RBC surface was assessed by gel agglutination test using a mouse monoclonal antibody specific to acridine. In vitro function was assayed pre- and 20 hours post-treatment, and weekly for 35 days (d) of storage. Parameters assessed for in vitro RBC function include intracellular ATP, 2,3-DPG, hemolysis, and extracellular potassium, glucose and lactate.Results: No agglutination was detected by gel column for mSRBC compared to 4+ readings for oSRBC indicating a reduction in S-303 bound to the RBC surface. While S-303 treatment results in higher initial ATP levels compared to pre-treatment, the mSRBC ATP level is maintained throughout 35d of storage. At 35d, oSRBC displayed glucose and lactate concentrations comparable to control RBC. In contrast, mSRBC had lower glucose and higher lactate concentrations than control RBC, consistent with observed higher ATP levels from increased glucose metabolism. After 35d of storage, hemolysis for oSRBC, Erythrosol mSRBC, AS-5 mSRBC, SAG-M mSRBC, and PAGGS-M mSRBC was comparable to controls whereas AS-3 mSRBC hemolysis was similar with the exception of 1 donor in AS-3 that showed a higher hemolysis level. Extracellular potassium levels were unaffected by oSRBC and mSRBC treatment.Conclusion: A modified PI process has been developed for RBC that significantly reduces S-303 bound to the RBC surface compared to the original process while retaining the critical RBC functions. The reduction in bound S-303 offers the potential for PI treatment for RBC without immunologic reactivity.ATP (μmol/g Hb)% HemolysisGATMean (SD)Mean (SD)oSRBCt=03.9 (0.2)0.1 (0.0)t=20h4.8 (0.3)0.2 (0.0)4+35 day2.6 (0.4)0.4 (0.0)mSRBC Erythrosolt=03.4 (0.2)0.1 (0.0)t=20h5.7 (0.1)0.1 (0.0)035 day3.8 (0.2)0.1 (0.0)mSRBC AS-3t=03.4 (0.5)0.2 (0.1)t=20h6.2 (0.4)0.3 (0.1)035 day3.7 (0.4)0.7 (0.8)mSRBC AS-5t=03.3 (0.2)0.1 (0.1)t=20h5.4 (0.7)0.3 (0.4)035 day3.3 (0.4)0.5 (0.3)mSRBC SAG-Mt=03.10.0t=20h5.00.0035 dayin progressin progressmSRBC PAGGS-Mt=03.10.0t=20h5.10.0035 dayin progressin progress

Back-priming of the RCM1 leucocyte-reduction filter: consequences for filtration efficacy.

The RCM1 leucocyte-reduction filter has been incorporated into two types of blood-donation sets of which one uses back-priming of the leucocyte filter with SAG-M solution. In this study we have compared the leucocyte-reduction efficacy of the RCM1 filter with or without back priming with SAG-M solution. This study was a retrospective analysis of quality control data of 3529 leucocyte-filtered red cell units. Of these, 1002 whole-blood units were collected in a donation set that was back-primed by letting the SAG-M solution in the final storage bag run backwards through the filter into the bag of concentrated red cells. The remaining 2527 units were collected in a donation set that required no back-priming. Postfiltration leucocyte concentration was assessed using flow cytometry. There was a significant trend towards lower leucocyte concentration in the units in which back-priming of the filter preceded filtration, as compared to units filtered without back-priming [chi2trend= 18.8, degrees of freedom (d.f.) = 1, P < 0.0005]. Back-priming of the RCM1 filter for red cells with SAG-M solution is superior to no back-priming with regard to leucocyte-reduction efficacy.

A reply

We thank you for the opportunity to respond to Dr Horsey's comments on our case report and to clarify any ambiguity. The blood preparation given was indeed red cells in an SAGM preparation but Dr Horsey's calculations are somewhat misleading. Each SAGM unit contains around 220 ml red cells and 150 ml supernatant (100 ml SAGM anticoagulant plus 50 ml plasma). During storage, potassium leaks from the red cells (intracellular potassium concentration is approximately 100 mmol.l−1) down a concentration gradient into all components of the supernatant (plasma and SAGM solution not just the plasma component). Supernatant levels of around 30 mmol.l−1 can be expected after 3 weeks storage. We transfused 2 units (748 ml) of SAGM blood preparation; this amounts to a total potassium load of 9 mmol. In a 42-kg cachectic woman with a calculated blood volume of 2520 ml (plasma volume of around 1500 ml) this potassium load has the potential to raise the potassium level by 6 mmol.l−1. But this would assume that complete intravascular retention takes place, whereas it is known that potassium usually equilibrates rapidly between the plasma and the extracellular fluid. The laboratory potassium level of 9.5 mmol.l−1 and the ECG changes confirm that there was indeed significant intravascular retention. We appreciate that modest transfusions of this nature are usually safe and in our article tried to explore other avenues for such a marked effect. Some suggestions given in our paper were: endogenous potassium release (tumour manipulation), reduced circulating volume and cardiac output, rapid pressure bag infusion, etc. However, the timing of the cardiac arrest and clinical presentation would suggest the transfusion must be implicated. Anaesthetists should not be lulled into a false sense of security simply because modest volumes of blood preparations are being transfused.

赤血球Ｍ・Ａ・Ｐにおける大凝集塊形成の機構と形成防止法

Since 1993 the Japanese Red Cross has adopted a new additive solution for red cell concentrate (RC), MAP (mannitol-adenine-phosphate), which is a modifided SAGM solution. However, frequent macroaggregate formation in MAP-additive RC (MAP-RC) has become a problem. We investigated the mechanism of macroaggregate formation in MAP-RC and sought method to reduce the frequency of such formation. During 3-week storage at 4°C, macroaggegates were formed in 30% of the buffy coat-poor RC (BP-RC) prepared by the conventional method without MAP, but in 100% of MAP-RC.Electron microscopic observation of the macroaggregates showed that red cells were trapped in a network structure, and enzyme treatments indicated that the network was made of DNA that leaked from granulocytes. The granulocytes stored in the MAP solution, however, formed macroaggregates only if plasma was added to the cell-suspension medium. Immunoblotting analysis indicated that fibrinogen and fibronectin were the constituents of the macroaggregate in addition to the DNA. Pre-storage filtration of MAP-RC with a leukocyte-removal filter (PALL BPF4) or preparation of BP-RC with the top & bottom system completely prevented the macroaggregate formation in the MAP-RC. These results indicated that reduction of leukocytes in MAP-RC to less than 1×108/bag is necessary to prevent macroaggregate formation.

Distribution of blood gases, glucose and lactate within stored blood units.

The distribution of respiratory gases, hydrogen ions, glucose and lactate were studied in red cells suspended in SAGM solution and stored undisturbed at +4 degrees C in plastic (PVC) bags. After 42 days' storage there were considerable concentration differences between the supernatant and the red cell layer, as well as concentration gradients within the latter. Thus, pO2, sO2, pH and the glucose concentration were higher in the supernatant and pCO2 and the lactate concentration were higher in the red cell layer. These findings show that sampling from the mixed content of blood which has been stored sedimented in plastic containers will not be representative for the erythrocyte environment at the end of shelf life. This is of importance in attempts to understand storage effects on red cells.

II. Relation between Cellular Morphology and Viability

Red cells collected in CPD, suspended in SAGM solution and stored for 42 days at +4 degrees C, were tested before and after incubation for 1 h at 37 degrees C with respect to autologous 24-hour posttransfusion red cell survival, morphology, fluidity at low shear rate and adenylate content. Warming and to some extent chromium labelling of the cells resulted in improvement of the morphology and fluidity but in no increase in adenosine triphosphate, total adenylate concentration or posttransfusion survival. The conclusion is drawn that short-term improvement of the morphology before transfusion does not result in increased viability.

What is the 'optimum' optimal additive solution?

Spontaneous formation of clotted material occurs occasionally in all stored red cell units, but larger amounts have been noted in SAG-M optimal additive suspensions. These can be substantially reduced by substitution of the SAG-M solution by a SAG-M/CPDA-1 mixture suggesting that there is insufficient citrate in SAG-M red cell suspensions to completely inhibit coagulation of the residual plasma.

Storage of red cells in a CPD/SAGM system using Teruflex PVC.

A quadruple plastic bag system made of Teruflex PVC (Terumo) was tested. Red cells separated from plasma and buffy coat were resuspended in SAGM solution. After storage for 42 days, the 24-hour posttransfusion autologous survival was 73.3 +/- 6.6% (means +/- SD), range 61.8-80.7%, n = 10. The shape of the cells was changed so that normal or slightly abnormal morphology was found in 37 +/- 7% of the cells after 42 days. Spontaneous in vitro hemolysis was 0.63 +/- 0.32% (range 0.28-1.13). A sufficient glucose reserve still remained at the end of storage, but glucose consumption and lactate production were impaired during the last 2 weeks due to the increasing acidity. By extrapolation from other studies it is suggested that 35 days is a more suitable shelf life than 42 days in this system.

What is the ‘Optimum’ Optimal Additive Solution?

Spontaneous formation of clotted material occurs occasionally in all stored red cell units, but larger amounts have been noted in SAG-M optimal additive suspensions. These can be substantially reduced by substitution of the SAG-M solution by a SAGM/ CPDA-1 mixture suggesting that there is insufficient citrate in SAG-M red cell suspensions to completely inhibit coagulation of the residual plasma.