Raman spectroscopy of stored red blood cell concentrate within sealed transfusion blood bags.

The standard practice in blood banks worldwide involves storage of red blood cells (RBCs) in plastic bags until they are needed for transfusion. During storage, the cells gradually degrade in functionality, a condition described as RBC storage lesion. Standard analytical techniques cannot assess the blood quality without breaching the sterility of the transfusion bag. In this study, we employed a commercially available spatially offset Raman spectroscopy (SORS) system using a custom designed protocol to non-invasively explore the biochemical changes in RBC concentrate of healthy donors over a storage period of approximately 42 days in standard transfusion bags, under standard storage conditions. The results reveal an increase in the oxygenation state of haemoglobin over the storage period for all donors, but different profiles for each donor. This study demonstrates the feasibility of acquiring consistent biochemical information relevant to the quality of stored blood, in situ through sealed blood transfusion bags using a commercially available instrument.

Alternatives in blood operations when choosing non-DEHP bags.

The use of di-ethylhexyl-phthalate (DEHP) in blood bags is under discussion due to toxicity concerns and possible restrictions. A questionnaire among 15 blood centres in nine countries showed that none so far have fully switched to non-DEHP blood bags. If centres had to change, sites with a 42-day outdate would choose for a shorter outdating period, while others would allow a higher haemolysis rate (but within current specifications). To improve red cell quality, about half of the centres are willing to move to an alternative red cell storage solution, while the other half would not change for various reasons.

Early γ-irradiation and subsequent storage of red cells in SAG-M additive solution potentiate energy imbalance, microvesiculation and susceptibility to stress-induced apoptotic cell death.

γ-Irradiation of red blood cell (RBC) concentrates prevents transfusion-associated graft-versus-host disease but may diminish RBC quality. Herein, we show that early γ-irradiation (25 Gy) of RBC units and their subsequent storage in SAG-M additive solution altered membrane microvesiculation, supernatant haemoglobin and cytosolic ATP. γ-Irradiation did not influence phosphatidylserine externalization, a marker of erythrocyte apoptotic cell death (eryptosis), in RBC stored for 42 days. However, shorter periods (4-21 days) of storage accentuated eryptosis in γ-irradiated RBC versus untreated RBCs following energy depletion, suggesting that γ-irradiated RBC is primed for stress-induced eryptosis during storage.

Experiences with semi-routine production of riboflavin and UV-B pathogen-inactivated platelet concentrates in three blood centres.

BACKGROUND: For a clinical platelet (PLT) transfusion trial conducted in three countries, the production of PLT concentrates (PCs) that were pathogen inactivated with the Mirasol technology was set up and validated. While the Mirasol procedure is applied to an established PLT product, the PLT processing procedure still had to be modified to ensure a treated PC was of sufficient quality. Further, the effect of simulated transport conditions and the effect of ambient light on Mirasol-treated PCs was determined. STUDY DESIGN AND METHODS: Platelet concentrates in plasma were made from pooled buffy coats followed by Mirasol treatment. To mimic transport conditions, units were left unagitated for 6 h at room temperature. To mimic ambient light exposure, units were held unagitated for 4 h in direct fluorescent tube light. RESULTS: Measures had to be taken to allow 7-day storage of treated concentrates. In one site, PCs made from five buffy coats with >450 × 109 PLTs were removed from inventory. Another site went from five to four buffy coats per pool. Interruption of agitation for 6 h on day 3 did not induce meaningful changes in in vitro measures, even when stored up to 7 days. Exposure to ambient light for 4 h, either on day 3 or 6, had no effect on in vitro measures. CONCLUSION: The Mirasol pathogen inactivation process can be implemented in routine, but changes to current PLT processing methods might be needed. Transport conditions and 4-h-long ambient light exposure have no negative effect on the in vitro quality of Mirasol-treated PCs.

An investigation of red blood cell concentrate quality during storage in paediatric-sized polyvinylchloride bags plasticized with alternatives to di-2-ethylhexyl phthalate (DEHP).

BACKGROUND AND OBJECTIVES: Di-2-ethylhexyl phthalate (DEHP) is a blood bag plasticizer. It is also a toxin, raising concerns for vulnerable populations, for example, neonates and infants. Here, the in vitro quality of red cell concentrates (RCC) stored in paediatric bags formulated with alternative plasticizers to DEHP was compared. MATERIALS AND METHODS: RCC were pooled and split into polyvinylchloride (PVC)/DEHP, PVC/1,2-cyclohexanedicarboxylic acid diisononyl ester (DINCH) or PVC/butyryl trihexyl citrate (BTHC) bags. Quality was assessed on storage days 5, 21, 35 and 43. RESULTS: Metabolism differed among the bags: pCO2 levels were lowest and pO2 were highest in BTHC bags. Glucose consumption and lactate production suggested higher metabolic rates in BTHC bags. ATP levels were best maintained in DINCH bags (day 43 mean level: 2·86 ± 0·29 µmol/g Hb). RCC in BTHC bags had the greatest potassium release (54·6 ± 3·0 mm on day 43). From day 21, haemolysis was higher in BTHC bags (P < 0·01) and by day 43 had exceeded 0·8% (0·85 ± 0·10%). RCC in BTHC bags showed more microparticle formation than RCC in DEHP or DINCH bags. CONCLUSION: The results suggest that the BTHC formulation used was detrimental to RBC quality. DINCH bags could be a viable alternative to DEHP: they outperformed DEHP bags energetically, with better maintenance of ATP levels.

Non-invasive spectroscopy of transfusable red blood cells stored inside sealed plastic blood-bags.

After being separated from (donated) whole blood, red blood cells are suspended in specially formulated additive solutions and stored (at 4 °C) in polyvinyl chloride (PVC) blood-bags until they are needed for transfusion. With time, the prepared red cell concentrate (RCC) is known to undergo biochemical changes that lower effectiveness of the transfusion, and thus regulations are in place that limit the storage period to 42 days. At present, RCC is not subjected to analytical testing prior to transfusion. In this study, we use Spatially Offset Raman Spectroscopy (SORS) to probe, non-invasively, the biochemistry of RCC inside sealed blood-bags. The retrieved spectra compare well with conventional Raman spectra (of sampled aliquots) and are dominated by features associated with hemoglobin. In addition to the analytical demonstration that SORS can be used to retrieve RCC spectra from standard clinical blood-bags without breaking the sterility of the system, the data reveal interesting detail about the oxygenation-state of the stored cells themselves, namely that some blood-bags unexpectedly contain measurable amounts of deoxygenated hemoglobin after weeks of storage. The demonstration that chemical information can be obtained non-invasively using spectroscopy will enable new studies of RCC degeneration, and points the way to a Raman-based instrument for quality-control in a blood-bank or hospital setting.

A clinical governance framework for blood services.

BACKGROUND AND OBJECTIVES: The elements of clinical governance, which ensure excellence in clinical care, can be applied to blood services. In this survey, their application in a range of blood providers was gauged, with the aim of identifying best practice and producing a generalizable framework. MATERIALS AND METHODS: The Medical Directors of members of the Alliance of Blood Operators surveyed how different elements of clinical governance operated within their organizations and developed recommendations applicable in the blood service environment. RESULTS: The recommendations that emerged highlighted the importance of an organization's culture, with the delivery of optimal clinical governance being a corporate responsibility. Senior management must agree and promote a set of values to ensure that the system operates with the patient and donor at its heart. All staff should understand how their role fits into the 'journey to the patient', and a culture of openness promoted. Thus, reporting of errors and risks should be actively sought and praised, with penalties applied for concealment. Systems should exist to collect, analyse and escalate clinical outcomes, safety data, clinical risk assessments, incident reports and complaints to inform organizational learning. CONCLUSION: Clinical governance principles from general health care can be applied within blood services to complement good manufacturing practice. This requires leadership, accountability, an open culture and a drive for continuous improvement and excellence in clinical care.

The effect of processing method on the in vitro characteristics of red blood cell products.

BACKGROUND AND OBJECTIVES: While the clinical impact of differences in red blood cell (RBC) component processing methods is unknown, there are concerns they may be confounding variables in studies such as the ongoing 'age of blood' investigations. Here, we compare the in vitro characteristics of red cell concentrates (RCCs) produced by several different processing methods. MATERIALS AND METHODS: Nine processing methods were examined: three apheresis methods (Alyx, MCS+ and Trima), as well as leucoreduced whole blood-derived RCCs produced by buffy coat and whole blood filtration and non-leucoreduced RCCs. RCCs were stored in saline-adenine-glucose-mannitol or additive solutions (AS) 1 or 3 for 42 days, with quality tested on day 5 and day 42. RESULTS: Many significant product differences were observed both early in and at the end of storage. Mean haemoglobin (Hb) ranged from 52 to 71 g/unit and mean Hct from 59·5 to 64·8%. Most RCC passed regulated quality control criteria according to Canadian Standards Association guidelines, although there were some failures relating to Hb content and residual WBC counts. CONCLUSION: Processing method impacts RCC characteristics throughout storage; better understanding of these differences and reporting of processing method details is critical.

The effect of timing of gamma-irradiation on hemolysis and potassium release in leukoreduced red cell concentrates stored in SAGM.

While irradiation of red cell concentrates (RCC) prevents graft-versus-host disease in susceptible transfusion recipients, it also damages red blood cells (RBC). To understand the ability of irradiation regulations to prevent transfusion of inferior units, we irradiated 980 RCC in saline-adenine-glucose-mannitol (SAGM) using various combinations of pre-irradiation age and post-irradiation storage times, and measured hemolysis and extracellular potassium levels. We observed unacceptably high hemolysis (>0·8%) in some RCC and elevated extracellular potassium levels in all gamma-irradiated RCC. This suggests that more restrictive storage times should be considered for RCC in SAGM.

Standardization of CD62P measurement: results of an international comparative study.

BACKGROUND AND OBJECTIVES: Despite long being a mainstay in describing platelet activation via degranulation, interlaboratory variation remains an issue in measurement of membrane CD62P by flow cytometry. Our objective was to identify actions that may minimize this variation. MATERIALS AND METHODS: Sixteen laboratories participated in an international comparative study. Two sets of platelet samples were prepared in one laboratory. Set 1 was stained and fixed; set 2 was fixed and required staining at participating laboratories. A single-staining method was used, and platelet populations were selected based on forward scatter/side scatter characteristics. Calibration beads were used to standardize measurement across different instruments. RESULTS: There was a large discrepancy in reported CD62P values among study sites [interlaboratory coefficient of variance (CV): 36-78%]. When electronic data were re-analysed by a single analyst using a consistent gating strategy and a stable reference point, variation decreased markedly (CV < 12%), indicating a problem with isotype control samples, possibly related to sample fixation or shipment. CONCLUSION: Consensus regarding gating strategies and use of a reliable reference point would greatly improve agreement in interlaboratory CD62P measurement.

Application of the ADVIA cerebrospinal fluid assay to count residual red blood cells in blood components.

BACKGROUND AND OBJECTIVES: There is no automated, accurate assay for the enumeration of residual red blood cells (rRBCs) in non-RBC components for transfusion, despite the potential risk of allo-immunization when mismatched components are transfused. MATERIALS AND METHODS: The automated ADVIA 120 cerebrospinal fluid (CSF) assay, which is approved to count RBCs and WBCs in CSF samples, was optimized and tested to measure rRBC in platelet concentrate (PC) and plasma components. RESULTS: Sample dilution, incubation time and reagent volume were optimized for use with non-RBC blood products. The assay was linear (R(2) = 0·99), even at low rRBCs counts. Intra- and inter-assay variation gave coefficients of variance (CV) between 2·2 and 9·4% and 2·6 and 14·9%, respectively, depending on rRBC levels. Good correlation (r = 0·995) was found between the automated assay and manual counting, which is considered the gold standard. Using the automated assay, the range of rRBCs (count/unit) in buffy-coat platelet concentrate (PCs) was 27-5505 × 10(6) and in apheresis PCs was 1-361 × 10(6). CONCLUSION: The ADVIA CSF assay is a sensitive, precise and accurate means to assess rRBC counts in non-RBC components.

Proteomic applications in blood transfusion: working the jigsaw puzzle.

The application of proteomic technologies to transfusion medicine has opened new avenues to our understanding of the products we prepare for patients and the processes that impact the quality of those products. The development of the field of proteomics has paralleled that of transfusion medicine with over a century of key scientific accomplishments required to bring us to our modern systems. We review the technology of proteomics and its application to transfusion medicine with specific reference to the analysis of blood products, both fractionated and fresh. Although the use of proteomic tools to address transfusion medicine questions is really just beginning, it is clear that this method of analysis provides different insights into unaddressed issues in the area of blood product research. Proteomics also offers the promise of improving our approach to the control of blood product quality and even the assessment of blood donors, but these are efforts for the near future.

De novo protein synthesis in mature platelets: a consideration for transfusion medicine.

Platelet function in thrombosis and haemostasis is reasonably well understood at the molecular level with respect to the proteins involved in cellular structure, signalling networks and platelet interaction with clotting factors and other cells. However, the natural history of these proteins has only recently garnered the attention of platelet researchers. De novo protein synthesis in platelets was discovered 40 years ago; however, it was generally dismissed as merely an interesting minor phenomenon until studies over the past few years renewed interest in this aspect of platelet proteins. It is now accepted that anucleate platelets not only have the potential to synthesize proteins, but this capacity seems to be required to fulfil their function. With translational control as the primary mode of regulation, platelets are able to express biologically relevant gene products in a timely and signal-dependent manner. Platelet protein synthesis during storage of platelet concentrates is a nascent area of research. Protein synthesis does occur, although not for all proteins found in the platelet protein profile. Furthermore, mRNA appears to be well preserved under standard storage conditions. Although its significance is not yet understood, the ability to replace proteins may form a type of cellular repair mechanism during storage. Disruption by inappropriate storage conditions or processes that block protein synthesis such as pathogen reduction technologies may have direct effects on the ability of platelets to synthesize proteins during storage.

A comparative study of common techniques used to measure haemolysis in stored red cell concentrates.

BACKGROUND AND OBJECTIVES: There is no standardized method of measuring the parameters for haemolysis determination of red cell concentrate (RCC). Three haemoglobin quantification methods (automated analyser, Harboe and Drabkin's) and two methods of haematocrit measurement (automated analyser and microcapillary centrifugation) were evaluated for use with RCC. MATERIALS AND METHODS: Twenty stored RCC were assayed for total haemoglobin, supernatant haemoglobin and haematocrit. RESULTS: Drabkin's and Harboe methods were linear (r(2) > or = 0.995) over 0.015-220 g/l haemoglobin. Overestimation by Drabkin's increased from 0% at 220 g/l to 137% at 0.015 g/l haemoglobin. Harboe values generally stayed within 6% of expected while haematology analyser values had a maximum 11% underestimation above 10 g/l. Analyser total haemoglobin was significantly lower (202 +/- 22 g/l) than Drabkin's (224 +/- 24 g/l) and Harboe (222 +/- 22 g/l) values. Haematocrit was greater via the analyser (65.7 +/- 5.7%) than with microcapillary centrifugation (59.3 +/- 5.7%). CONCLUSIONS: Harboe and Drabkin's methods are suitable for measuring total haemoglobin and supernatant haemoglobin in RCC. The analyser gave higher haematocrit values (11% on average) than did microcapillary centrifugation.

Inventory management.

A critical aspect of blood transfusion is the timely provision of high quality blood products. This task remains a significant challenge for many blood services and blood systems reflecting the difficulty of balancing the recruitment of sufficient donors, the optimal utilization of the donor's gift, the increasing safety related restrictions on blood donation, a growing menu of specialized blood products and an ever-growing imperative to increase the efficiency of blood product provision from a cost perspective. As our industry now faces questions about our standard practices including whether or not the age of blood has a negative impact on recipients, it is timely to take a look at our collective inventory management practices. This International Forum represents an effort to get a snap shot of inventory management practices around the world, and to understand the range of different products provided for patients. In addition to sharing current inventory management practices, this Forum is intended to foster an exchange of ideas around where we see our field moving with respect to various issues including specialty products, new technologies, and reducing recipient risk from blood transfusion products.

Comparison of a novel viscous platelet additive solution and plasma: preparation and in vitro storage parameters of buffy-coat-derived platelet concentrates.

BACKGROUND AND OBJECTIVES: We developed a viscous platelet additive solution (PAS) based on MacoPharma's SSP+ but containing hydroxyethyl starch to address the poor osmotic balance and low yield associated with conventional PAS for the storage of buffy-coat platelet concentrates (PC). MATERIALS AND METHODS: Pools of four buffy-coats were made into leucoreduced PCs (n = 5) suspended either in plasma or viscous PAS. After determination of platelet recoveries, the PCs were stored under standard conditions. On days 1, 2, 3, 5, 7 and 9, PCs were tested for mean platelet volume, platelet concentration, soluble protein concentration, CD62 expression, platelet morphology, partial pressure of oxygen and partial pressure of carbon dioxide, glucose and lactate concentration, pH, extent of shape change, and hypotonic shock response (HSR). RESULTS: Platelets were prepared with greater ease using the viscous PAS and had improved platelet yield. PCs stored in either plasma or viscous PAS displayed similar storage characteristics to day 9. On days 7 and 9 of storage, platelets stored in viscous PAS displayed significantly lower (P < 0.05) CD62 expression and higher HSR scores than those stored in plasma. CONCLUSION: Alteration of the viscosity of PAS improves platelet recovery during processing and may prolong platelet quality at the later stages of storage.

Interactions of the platelets in paroxysmal nocturnal hemoglobinuria with complement. Relationship to defects in the regulation of complement and to platelet survival in vivo.

The blood cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) have abnormal interactions with complement. The activity of the alternative pathway C3 convertase on the platelets of 9 out of 19 patients with PNH was elevated. 10 patients had C3 convertase activity within the normal range even though 80-95% of their platelets lacked the complement regulatory protein decay accelerating factor (DAF) that is absent from the affected blood cells in PNH. PNH and normal platelets released factor H when C3 was bound to their surfaces. This may account for the apparent regulation of C3 convertase activity on platelets that lack DAF. The abnormal uptake of the membrane attack complex of complement by PNH III erythrocytes was not seen in PNH platelets. 111Indium-labeled platelet survival times were normal in five of eight patients, which suggests that the lack of the membrane attack complex defect results in normal platelet survival in PNH.

Reactions of immunoglobulin G-binding ligands with platelets and platelet-associated immunoglobulin G.

Immunoglobulin G (IgG) bound to platelets is usually detected by one of two general methods: binding of labeled anti-IgG or consumption of anti-IgG. The latter method gives, in general, values 5-10-fold greater than the former under the same conditions. To investigate these discrepancies, we have compared the detection of platelet-bound IgG by a labeled anti-IgG binding assay and by a quantitative antiglobulin consumption test using the same antibodies. The interaction of 125I-labeled monoclonal anti-IgG or polyclonal anti-IgG with washed and IgG-coated platelets was studied. The binding of these ligands to washed normal platelets was largely (50-80%) nonspecific; the binding was not saturable and was only partially inhibitable by excess unlabeled anti-IgG. The binding of anti-IgG to platelets coated with anti-PIA1, a platelet-specific IgG antibody, appeared to be saturable and inhibitable; the dissociation constant (KD) of this IgG-anti-IgG reaction was 4.9 X 10(-9) for monoclonal and 1.4 X 10(-7) for polyclonal anti-IgG. The ratio of sites present on the membrane (determined by 131I-labeled anti-PIA1) to the number of binding sites for anti-IgG determined by Scatchard analysis was 0.53 for monoclonal anti-IgG and 1.3 for polyclonal anti-IgG. The binding of monoclonal anti-IgG to platelet-bound immune complexes or IgG aggregates appeared to be complex. 131I-Labeled IgG was affixed to platelets and was detected by three tests: direct binding of radiolabeled monoclonal anti-IgG and quantitative antiglobulin consumption (QAC) tests, which were quantitated either by measuring directly the amount of radiolabeled anti-IgG consumed from fluid phase (direct QAC), or indirectly by reference to a calibration curve relating the consumption of anti-IgG by known amounts of fluid-phase, non-immune IgG (indirect QAC). The amount of platelet-bound IgG detected by the direct binding of 125I-labeled monoclonal anti-IgG and by the direct QAC approximated that known to be bound to the platelet. The results of the indirect QAC test were 10-fold greater. The discrepancy appears to be due to the fact that there is a difference between the IgG-anti-IgG interaction when IgG is bound to a platelet and when it is in solution or bound to plastic nonspecifically or specifically. This difference results in a falsely high value for platelet-bound IgG when fluid-phase or plastic-bound IgG is used to calibrate the antiglobulin consumption test.