Posttransfusion Survival Research Articles

Use of an in vitro biotinylation technique for determination of posttransfusion survival of fresh and stored autologous red blood cells in Thoroughbreds

To evaluate N-hydroxysuccinimide (NHS)-biotin labeling of equine RBCs and determine posttransfusion survival of autologous equine RBCs stored in citrate phosphate dextrose adenine-1 (CPDA-1) for 0, 1, 14, and 28 days. 13 healthy adult Thoroughbreds. Serial dilutions of biotin and streptavidin-phycoerythrin (PE) were evaluated in vitro in blood collected from 3 horses. One horse was used to determine RBC distribution and recovery. Twelve horses were allocated to 4 groups for in vivo experiments in which blood was collected into CPDA-1. Blood was labeled with biotin and reinfused or stored at 4 degrees C for 1, 14, or 28 days prior to labeling with NHS-biotin and reinfusion. Posttransfusion blood samples were collected 15 minutes and 1, 2, 3, 5, 7, 14, 21, 28, and 35 days after reinfusion. Biotin-labeled RBCs were detected via flow cytometry by use of streptavidin-PE. Posttransfusion lifespan of RBCs and RBC half-life were determined. Optimal biotin concentration was 0.04 pg of biotin/RBC, and the optimal streptavidin-PE ratio was 1.2 microg of streptavidin-PE/1 x 10(6) RBCs. Posttransfusion lifespan of autologous RBCs was 99, 89, 66, and 59 days after storage for 0, 1, 14, and 28 days, respectively. Storage did not result in significant alterations in RBC lifespan. Mean posttransfusion RBC half-life was 50, 45, 33, and 29 days for 0, 1, 14, and 28 days of storage, respectively. Biotin can be used to label equine RBCs for RBC survival studies. Posttransfusion survival of equine autologous RBCs was greater than previously reported.

An approach to prevent the severe adverse events associated with transfusion of FDA-approved blood products

There have been several retrospective studies reporting severe adverse events of mortality and morbidity associated with blood transfusions. Mortality and morbidity associated with posttransfusion infection, transfusion related acute lung injury (TRALI), and systemic inflammatory response syndrome (SIRS) have been reported in patients undergoing cardiac surgery, after massive transfusions for severe traumatic injuries, and after transfusions for elective and emergency indications. After 35 days of storage at 4 °C in additive solutions, RBC have 24-h posttransfusion survival values of 75% but do not function satisfactorily. For RBC to function satisfactorily shortly after transfusion, they should be stored at 4 °C for no more than 2 weeks. Yet while the FDA requires a 24-h posttransfusion survival value of 75%, there is no requirement for the function of the transfused RBC. It has been shown that red blood cells that circulate and function immediately or shortly after transfusion exert a very important hemostatic effect to reduce the bleeding time and nonsurgical blood loss in anemic and thrombocytopenic patients. Greater restoration of hemostasis is seen with viable and functional RBC transfusions than with platelets or plasma even though the platelets and plasma proteins may have satisfactory viability and function. The length of storage of the blood products affects their survival and function and the transfusion of nonviable compatible RBC, antibodies to granulocytes and WBC HLA antigens and biologically active substances affects the patient’s clinical outcome. One of the easiest ways to prevent the severe adverse events that have been observed is to ensure that the transfused blood products survive and function at an optimum level and that the levels of antibodies to granulocytes and WBC HLA antigens and biologically active substances are eliminated or reduced. The best way to ensure this is to store liquid-preserved leukoreduced human red blood cells at 4 °C in additive solutions for no more than 2 weeks and leukoreduced platelets at room temperature for no more than 2 days. These liquid-preserved blood products can be used in conjunction with frozen RBC, platelets, and plasma stored in −80 °C mechanical freezers and will avoid the need for fresh whole blood and prevent the severe adverse events associated with the transfusion of blood products.

P38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo

AbstractPlatelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-α and GPV. We recently demonstrated that tumor necrosis factor-α converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37°C or 22°C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

Leukoreduction Decreases Alloimmunogenicity of Transfused Murine HOD RBCs.

Abstract 640 Introduction:Alloimmunization against transfused red blood cell (RBC) antigens can lead to hemolytic transfusion reactions as well as difficulties in locating compatible units of blood for future transfusions. Despite the clinical significance, factors influencing rates of RBC alloimmunization are not clearly defined. Leukoreduction decreases rates of HLA alloimmunization, because HLA antigens are carried on white blood cells (WBCs). However, most human RBC antigens are not carried on WBCs, and the effect of leukoreduction on RBC alloimmunization is not known. Many potential variables, including the large number of RBC antigenic differences between donor and recipient, make this question difficult to answer in the setting of human transfusion medicine. We hypothesized that WBCs in transfused RBC units would increase alloimmunization to an RBC specific antigen, and used a reductionist murine model to investigate this issue. Materials and Methods:Transgenic HOD donors, with RBC specific expression of the model antigen hen egg lysozyme (HEL) linked to a multi-pass human Duffy antigen (Fyb), were bled into CPDA-1 to give a final storage medium concentration of 14%. Leukoreduction was performed over a Pall neonatal leukoreduction filter, with residual WBCs quantified by propridium iodide and Trucount beads. Expression of HEL and Fyb on HOD RBCs was assessed by flow cytometry prior to transfusion. Leukoreduced or non-leukoreduced pRBCs (75 μL) were transfused via lateral tail vein, and post-transfusion survival of HOD RBCs was determined by analysis of HEL and Fybexpression at 10 and 30 minutes, 2 and 24 hours following transfusion. Alloimmunization was assessed 2 weeks later by HEL-specific ELISA and flow cytometric crossmatching (using HOD or control FVB RBCs). Statistical significance (p<0.05) was determined using PRISM software and a two-way ANOVA with a Bonferroni post-test. Results:Alloantibodies against HOD RBCs were detectable by HEL-specific ELISA in recipients following a single transfusion of non-leukoreduced or leukoreduced RBCs. In 4 of 5 experiments (n=50 mice total), non-leukoreduced HOD RBCs were statistically significantly more immunogenic than leukoreduced HOD RBCs (sera diluted 1:50). Alloantibodies against HOD RBCs were detected in a minority of mice by flow cytometric crossmatching, which is less sensitive than ELISA. By histogram overlay of sera crossmatched with HOD RBCs compared to control FVB RBCs, 6/25 recipients of non-leukoreduced RBCs and 2/25 recipients of leukoreduced RBCs had a detectable anti-HOD response. In each experiment, there was a >3 log10 decrease in leukocytes after leukoreduction. HEL and Fyb expression was comparable as determined by flow cytometry in samples pre- and post-leukoreduction. Mean 24 hour post-transfusion survival was 98.7% (95% CI 97-100%) and 97.9% (95% CI 95.4-100%) in recipients of non-leukoreduced and leukoreduced RBCs, respectively. Conclusions:Leukoreduction of murine HOD RBCs decreases alloimmunogenicity of the RBC specific HEL antigen. This decrease is subtle and is better detected by HEL-specific ELISA than by less sensitive flow cytometric crossmatching. Although one explanation for these findings is that the WBCs themselves are contributing to the increased immunogenicity by providing a danger signal, it is also possible that non-specific alloreactivity of transfused WBCs or other cells is playing a role. Furthermore, it cannot be ruled out that changes in platelets or RBCs induced by the leukoreduction filter are involved. However, the observed changes in immunogenicity cannot be explained by a decrease in HEL expression post-leukoreduction or by altered clearance of leukroreduced RBCs after transfusion, as HEL and Fyb expression, as well as 24 hour post-transfusion survival of HOD RBCs, were similar in both groups. If the immunogenicity of other RBC antigens is also decreased after leukoreduction, then patients at highest risk of RBC alloimmunization may benefit from receiving solely leukoreduced blood products. Furthermore, the development of higher efficiency and/or modified leukoreduction filters may be warranted. Disclosures:No relevant conflicts of interest to declare.

Effects of pretransfusion warming of platelets to 35°C on posttransfusion platelet viability

Three of four prior studies suggested that warming platelets (PLTs) to 37 degrees C before transfusion into patients with thrombocytopenia gave improved corrected PLT count increments. Eighteen normal subjects had apheresis PLTs collected that were stored at 22 degrees C for 5 days in two storage bags. One bag of PLTs was warmed to 35 degrees C before infusion, and one remained at 22 degrees C. Three different methods of warming the donor's autologous PLTs before reinfusion were evaluated: warming PLTs to 35 degrees C for 10 or 60 minutes followed by radiolabeling or radiolabeling the PLTs followed by warming to 35 degrees C for 60 minutes. In the first two methods, the warmed PLTs would have returned to 22 degrees C before infusion, and in the third, the PLTs would still be warm when injected. The paired test and control PLTs were radiolabeled with either (111)In or (51)Cr to determine posttransfusion PLT recoveries and survivals. PLT morphology score, pH, hypotonic shock response, extent of shape change, and annexin V binding were determined just before transfusion. There were no differences in posttransfusion autologous radiolabeled PLT recoveries and survivals or in the in vitro measurements for the PLTs maintained at 22 degrees C versus those warmed to 35 degrees C using any of the three methods of PLT warming before infusion. Based on these 5-day-stored autologous radiolabeled PLT recovery and survival measurements, there is no evidence that warming PLTs to 35 degrees C before infusion improves postinfusion PLT viability.

A randomized study of buffy coat platelets in platelet additive solution stored 1–5 versus 6–7 days prior to prophylactic transfusion of allogeneic haematopoietic progenitor cell transplant recipients

Storage of platelets > 5 days provides improved availability, logistical management and decreased outdating. Promising results on in vitro parameters and on in vivo post-transfusion recovery and survival of autologous platelets in healthy volunteers have earlier been shown. To provide additional verification, randomized patient transfusion studies are needed. Sixty allogeneic haematopoietic progenitor cell transplant recipients were randomized to receive buffy-coat (BC) platelets stored in platelet additive solution (PAS) for 1-5 days the first time a prophylactic transfusion was needed after transplantation, followed the second time by platelets stored for 6-7 days or vice versa. The corrected count increment (CCI) for 1 and 24 h were calculated. CCI 1 h and CCI 24 h were higher for platelets stored 1-5 days as compared to 6-7 days, 10.4 +/- 5.1 vs. 7.4 +/- 3.8 (P < 0.001) and 5.4 +/- 4.1 vs. 2.6 +/- 2.6 (P < 0.001), respectively. Time to next platelet transfusion was significantly longer after a transfusion of platelets stored for 1-5 days as compared to platelets stored for 6-7 days: 2.2 +/- 1.1 vs. 1.6 +/- 0.8 days, respectively (P < 0.005). No differences in bleeding events and no transfusion reaction were recorded. The advantage of an extension of platelet storage time beyond day 5 should be balanced against the increased need for platelet transfusions that may occur and the conceivable risk of transfusion failure.

A novel mouse model of red blood cell storage and posttransfusion in vivo survival

Storage of red blood cells (RBCs) is necessary for an adequate blood supply. However, reports have identified potential negative sequelae of transfusing stored RBCs. An animal model would be useful to investigate the pathophysiology of transfusing stored RBCs. However, it has been reported that storage of rat RBCs in CPDA-1 resulted in an unexpected sudden decline in posttransfusion survival. A mouse model of RBC storage and transfusion was developed to assess survival kinetics of mouse RBCs. RBCs expressing green fluorescent protein were collected in CPDA-1, filter leukoreduced, adjusted to a 75% hematocrit, and stored at 4°C. At weekly intervals, stored RBCs were transfused into C57BL/6 recipients. RBC survival was measured by flow cytometry and chromium-51 labeling. Phosphatidylserine externalization and CD47 expression was also evaluated. Mean 24-hour survivals of transfused RBCs were 99, 91, 64, 54, 30, and 18% after 0, 7, 14, 21, 28, and 35 days of storage, respectively. Stored RBCs showed an initial rapid clearance with subsequent extended survival. Increased surface phosphatidylserine and decreased CD47 expression were also observed. Mouse RBCs showed a progressive decline in survival, as a function of storage time, unlike the precipitous loss of viability reported for rat RBCs. Moreover, changes in the measured surface markers were analogous to trends reported for human RBCs. Together, these findings provide an initial characterization of a novel mouse model of RBC storage with the potential to serve as an experimental platform for studying the pathophysiologic consequences of transfusing stored RBCs.

Post‐transfusion stability of haemoglobin mass

Total haemoglobin mass (tHb) as a direct parameter of the blood system and ultimate target of all blood transfusions has not been evaluated for its post-transfusion survival and stability. Therefore, the purpose of this study was to investigate the latter which may also be relevant from an anti-doping perspective as autologous blood transfusions remain impossible to detect. The tHb was determined by the CO rebreathing method prior to and after donation of 1 unit of whole blood, as well as prior to and after reinfusion (weekly up to 56 days) of the erythrocyte concentrate in 10 men (28 +/- 7 years, 181 +/- 7 cm, 76 +/- 12 kg). The mean tHb content of the derived erythrocyte concentrate was 60 +/- 3 g, while the net tHb increases after transfusion of 51 g (95% confidence intervals 33-69 g) permitted proof of an elevated tHb for at least 56 days after transfusion. The results show that an elevated tHb induced by autologous transfusion allowed continuous identification although, as expected, a slow decrease of tHb has been revealed in the observation period. In reference to anti-doping, CO rebreathing permits proof of a supraphysiologically elevated tHb but possibly only if a stable baseline value is known.

Survival after blood transfusion

Long-term survival of transfusion recipients has rarely been studied. This study examines short- and long-term mortality among transfusion recipients and reports these as absolute rates and rates relative to the general population. Population-based cohort study of transfusion recipients in Denmark and Sweden followed for up to 20 years after their first blood transfusion. Main outcome measure was all-cause mortality. A total of 1,118,261 transfusion recipients were identified, of whom 62.0 percent were aged 65 years or older at the time of their first registered transfusion. Three months after the first transfusion, 84.3 percent of recipients were alive. One-, 5-, and 20-year posttransfusion survival was 73.7, 53.4, and 27.0 percent, respectively. Survival was slightly poorer in men than in women, decreased with increasing age, and was worst for recipients transfused at departments of internal medicine. The first 3 months after the first transfusion, the standardized mortality ratio (SMR) was 17.6 times higher in transfusion recipients than in the general population. One to 4 years after first transfusion, the SMR was 2.1 and even after 17 years the SMR remained significantly 1.3-fold increased. The survival and relative mortality patterns among blood transfusion recipients were characterized with unprecedented detail and precision. Our results are relevant to assessments of the consequences of possible transfusion-transmitted disease as well as for cost-benefit estimation of new blood safety interventions.

Platelet Transfusion - Back to Basics

As in many aspects of transfusion medicine, practices have developed based on low levels of evidence. Let’s review some assumptions and think how these might change in future:- (1) Indications. 80% of platelet transfusions are given prophylactically to patients with bone marrow failure, yet no trials have demonstrated the value of prophylaxis over treatment of bleeding alone. (2) Trigger. A trigger of 10 × 10 g/l is now accepted. Reducing this further will depend on accurate counting, challenging at this level. (3) Dose & frequency. The optimum dosing schedule has not been derived by pharmacokinetic studies, but by what can be conveniently manufactured. An adult dose of 3 × 1011 platelets is accepted, but there is wide variation in practice. (4) Manufacture. Globally, 3 methods (pooled buffy coats, platelet rich plasma and apheresis) all produce an efficacious product. Although apheresis platelets reduce donor exposure, no functional advantage has been demonstrated. (5) In vitro assessment of function. A battery of tests is recommended, but only pH has been shown to correlate with posttransfusion survival. (6) ABO matching. ABO compatible platelets give better increments, and transfusing ABO incompatible plasma can be associated with haemolysis, but ABO matching to patient is often compromised by stock management. (7) Use of platelet additive solution. A 70:30 mix of PAS:plasma would reduce reactions and perhaps TRALI but this is not standard practice. Is the plasma useful? (8) CMV safety. Overall, leucocyte depletion alone appears to provide a high degree of protection, but this is not universally accepted. (9) Assessment of response. 1 and 24 hr increments are standard, but is regression analysis the way forward? (10) Management of refractoriness. Better HLA and HPA matching is now technically possible. Should these lead to better HLA matching for all? (11) Is one product for all appropriate? 20% of platelets go to surgical patients who are bleeding. Do they need a product with a different specification?

Long‐term survival rate of pediatric patients after blood transfusion

Studies in adults report posttransfusion survival rate (PTSR) at 1 to 10 years of 41 to 67 percent. There are no published studies specifically addressing PTSR in pediatric patients. The objectives of this study were to evaluate PTSR and risk factors associated with death in children receiving transfusions. A database of all patients receiving their first transfusion between 1990 and 1992 at Sainte-Justine Hospital (SJH) was used. Patients' demographic data, primary diagnosis, surgical procedures, and information on all labile components transfused were collected. Death was confirmed by the SJH database, RAMQ (universal health care provider for Québec residents), and the Quebec Commission for Access to Information (death certificate). The study population consisted of 1100 children. Median age was 16 months (range, 0-204 months). The most frequent primary diagnoses were cardiac disease (22%), prematurity (21.5%), malignant disease (11%), and hematologic disease (9.5%). Patients received a median of three transfusions (range, 1-126). PTSR was 86.9 percent at 1 year and 82.3 percent at 10 years. For nonsurvivors, median survival time was 22 days. Multivariate analysis showed that significant risk factors for death are age of less than 1 month, a diagnosis of malignant disease, more than 20 transfusions, and administration of more than one type of blood component. PTSR in children receiving their first transfusion between 1990 and 1992 was considerably higher than the PTSR reported in adults. Death occurred early after the first transfusion; survival rate remained relatively stable thereafter.

The Effects of Preserved Red Blood Cells on the Severe Adverse Events Observed in Patients Infused with Hemoglobin Based Oxygen Carriers

The severe adverse events observed in patients who received hemoglobin based oxygen carriers (HBOCs) were associated with the Ringer's D.L lactate resuscitative solution administered and to the excipient used in the HBOCs containing Ringer's D,L lactate and the length of storage of the preserved RBC administered to the patient at the time that the HBOCs were infused. This paper reports the quality of the red blood cells preserved in the liquid state at 4 degrees C and that of previously frozen RBCs stored at 4 degrees C with regard to their survival, function and safety. Severe adverse events have been observed related to the length of storage of the liquid preserved RBC stored at 4 degrees C prior to transfusion. The current methods to preserve RBC in the liquid state in additive solutions at 4 degrees C maintain their survival and function for only 2 weeks. The freezing of red blood cells with 40% W/V glycerol and storage at -80 degrees C allows for storage at -80 degrees C for 10 years and following thawing, deglycerolization and storage at 4 degrees C in the additive solution (AS-3, Nutricel) for 2 weeks with acceptable 24 hour posttransfusion survival, less than 1% hemolysis, and moderately impaired oxygen transport function with no associated adverse events. Frozen deglycerolized RBCs are leukoreduced and contain less than 5% of residual plasma and non-plasma substances. Frozen deglycerolized RBCs are the ideal RBC product to transfuse patients receiving HBOCs.

The Post-Transfusion Recovery and Survival of Red Blood Cells in Mice Is Affected by the Expression of Cellular Prion Protein.

Three documented transfusion cases of vCJD underline the need of better insight in blood prion protein biology. Cellular prion protein (PrPc) plays key role in the pathophysiology of prion diseases. Its expression by cells is necessary for amplification of infectious prions and the disease process itself. Physiological function of PrPc remains obscure. Its clarification may provide important clues for the development of urgently needed blood test and effective disease treatment. PrPc is expressed on CD34+ hematopoietic stem cells and its expression is regulated during blood cell differentiation. Recently the importance of PrPc for self-renewal of long-term repopulating hematopoietic stem cells was suggested and other studies reported the protective function of PrPc against oxidative stress and apoptosis in various cell cultures. We previously demonstrated that human as well as mouse red blood cells (RBC) express approximately 200 PrPc molecules / cell (Holada et al., BJH 2000, 110, 472–80). To test if the PrPc expression plays a role in the post-transfusion recovery and survival of RBC we carried out transfusion study in mice. RBC isolated from blood of wild type (WT) and PrP knockout (KO) FVB mice were labeled “in vitro” by different levels of NHS-biotin. The labeling was optimized to allow simultaneous detection of both populations of RBC in mouse blood using flow cytometry. To exclude the influence of different level of cell biotinylation on the experiment outcome two mixtures of RBC were prepared. The first contained KO RBC labeled with high and WT RBC with low level of biotin and the second mixture contained cells labeled “vice versa”. Each mixture was injected via tail vein in a group of WT mice (n=5) and the survival of RBCs was followed. Samples were analyzed on day 1, 2, 3, 6, 9, 15, 21 and 29. The count of biotinylated RBC was measured in comparison to 100 000 nonlabeled recipient RBC. Simultaneously the expression of PrPc on RBC was monitored using flow cytometry with MAb 6H4. KO RBC displayed significantly higher first day post-transfusion recovery compared to WT RBC in both groups of mice (81 ± 3 % vs. 74 ± 3 %, P<0.005 and 90 ± 4 % vs. 80 ± 4 %, P<0.005). The slope of the RBC survival curve in all individual mice during the initial 15 days was steeper for KO RBC (mavg = − 3.44) than for WT RBC (mavg = − 2.37) suggesting the protective role of PrPc in circulating RBC. The difference in the slope diminished during the 15 to 29 day period which was accompanied by a 50% decrease of PrPc surface expression on transfused WT RBC. To confirm our data the identical experiment was carried out in a group of KO mice (n=5) transfused with a mixture containing KO RBC labeled with low and WT RBC with high level of biotin. Again the first day post-transfusion recovery was higher for KO RBC (80 ± 6 % vs. 75 ± 6 %, P<0.05) and the initial slope of the KO RBC survival curve was steeper in all mice in the group. Our data suggest that PrPc expression plays role in the post-transfusion recovery and survival of RBC. The observation that WT RBC disappear from the circulation at lower rate than KO RBC until their level of surface PrPc reaches 50% is compatible with the protective role of PrPc expression on cells. Taken together our study demonstrates that physiological role of PrPc expression on RBC may lay in facilitating their longer survival in circulation. (GACR 310/04/0419, MSMT 0021620806).

Extended storage of red blood cells under anaerobic conditions

Red blood cells (RBC) are subject to oxidative stress by reactive oxygen species during refrigerated storage. Near-complete removal of oxygen from red cells during storage should eliminate this contributor to the red cell 'storage lesion'. The in vitro effects of storing red cells under oxygen-depleted conditions for extended periods were investigated, and these were correlated with the observed recoveries after reinfusion. Units of red cells, obtained after 'soft spin', were placed in a double volume of AS-3 additive solution and subdivided. Oxygen in the test units was depleted by repeated exposure to Ar gas (to O(2) saturation < 4%), and units were stored in anaerobic canisters for up to 15 weeks. Samples were taken weekly to monitor adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG), cell-free haemoglobin, and vesicle production. In a parallel experiment, six units of red cells was depleted of oxygen in a similar manner, stored for 8, 9 and 10 weeks, and reinfused autologously to determine the 24 h post-transfusion recovery via (51)Cr/(99m)Tc radiolabelling. A similar study was also carried out using EAS61 additive solution, which by itself, had shown the ability to support 9-week storage, comparing biochemical profiles and in vivo recovery after aerobic vs. anaerobic storage. Oxygen-depleted AS-3 units had significantly elevated ATP levels compared to controls. They also had significantly lower cell free haemoglobin and vesicle production when RBCs were stored for more than 9 weeks. An average of over 75% post-transfusion survival was observed after 9 weeks of anaerobic storage with less than 0.43% haemolysis. However, no further extension of storage was achieved with EAS61 additive. Anaerobic conditions permit acceptable 9-week storage of RBCs using double-volume AS-3 additive solution. It did not synergize with the alkaline, 9-week additive, EAS61, to further lengthen the acceptable storage time. These studies indicate that anaerobic storage may allow reduction in the effect of the storage lesion, but suggest that other factors contribute to limitations of RBC storage as well.

Viability and function of 8‐day‐stored apheresis platelets

Methods of bacterial detection and pathogen inactivation of platelets (PLTs) may allow extended storage of PLTs as long as PLT quality is maintained. Twenty normal volunteers had their PLTs collected with an apheresis machine (Haemonetics Corp.). A variety of in vitro PLT function and metabolic assays were performed both on Day 0 and after 8 days of storage. On Day 8, a small blood sample was drawn from each donor to obtain fresh PLTs. The fresh and stored autologous PLTs were labeled with either (51)Cr or (111)In, and the radiolabeled PLTs were transfused. Posttransfusion serial blood samples were drawn to determine the relative posttransfusion recoveries and survivals of the fresh versus the stored PLTs. Although the in vitro assays showed some differences between the two trial sites, the results were generally within the ranges expected for fresh and stored PLTs. Overall, PLT recoveries averaged 66 +/- 16 percent versus 53 +/- 20 percent and survivals averaged 8.5 +/- 1.6 days versus 5.6 +/- 1.6 days, respectively, for fresh compared to 8-day-stored PLTs. There were no significant differences in the in vivo PLT data between the trial sites or based on the radiolabel used for the measurements. After 8 days of storage, the in vivo posttransfusion recovery and survival of autologous Haemonetics apheresis PLTs meet the proposed standards for poststorage PLT quality.

Effects of centrifugal and roller pumps on survival of autologous red cells in cardiopulmonary bypass surgery

Either a roller pump or a centrifugal pump can be used in the extracorporeal circuit during surgery with cardiopulmonary bypass. In this study, we assessed the effect of these two pumps on the 24-h post-transfusion survival values of autologous red blood cells (RBC). Fourteen male patients subjected to extracorporeal bypass procedures were studied. In seven patients, the autologous red cells were collected following the cardiopulmonary bypass procedure using the roller pump, and in seven patients, autologous red cells were collected following the cardiopulmonary procedure using the centrifugal pump. The 24-h post-transfusion survival values of the autologous RBC were measured using the 51 disodium chromate/99m technetium double isotope procedure. The effects of the extracorporeal bypass procedures using the roller pump and the centrifugal pump were also assessed by the measurements of hematocrit, platelet count, plasma hemoglobin, and serum lactate dehydrogenase levels. The 51 disodium chromate 24-h post-transfusion survival values of the autologous RBC were similar whether the roller pump or the centrifugal pump was used in the extracorporeal circulation, as were the hematocrit, platelet count, plasma hemoglobin and serum lactate dehydrogenase levels. The 24-h post-transfusion survival values of autologous RBC, measured by the 51 disodium chromate/99m technetium double isotope procedure, were not significantly different, whether the roller pump or the centrifugal pump was used in the extracorporeal circuit using membrane oxygenators during cardiopulmonary surgical procedures.

24-Hour survival of autotransfused red cells in elective aortic surgery: A comparison of two intra-operative autotransfusion systems

Twelve patients undergoing elective aortic surgery had intraoperative autotransfusion using either the Haemonetics Cell Saver or the Solcotrans device. The 24-h post-transfusion survival of the patients' preoperative red cells, labelled with indium-111, was compared with the survival of salvaged red cells labelled with chromium-51. A correction coefficient for the instability of the indium-111 label was calculated from the study of red cell survival in six healthy volunteers. There was no significant difference in the volume of circulating red cells measured using each isotope, indicating that extensive early destruction of the autotransfused red cells does not occur. The percentage survival of autotransfused red cells at 24 h was similar to that of the red cells that had not undergone salvage. Both devices were comparable with respect to the effects of cell salvage on the 24-h survival rate of autotransfused red cells.

Neonatal Alloimmune Thrombocytopenia (NATP) Associated with Maternal-Fetal Incompatibility for Blood Group B.

In most individuals, A and B blood group antigens are weakly expressed on platelets, allowing ABO incompatible platelets to be tolerated when transfused. However, in a minority of normal subjects (high expressers, H-Exp), platelets carry 10–20 times the usual number of A or B epitopes (up to 20,000/platelet, Blood 2000; 96:1574). Post-transfusion survival of incompatible H-Exp platelets has not been systematically studied. We recently encountered a family in which NATP in two infants appears to have been caused by maternal anti-B reacting with H-Exp fetal platelets inherited from a father with the group B H-Exp trait. The first two children (C1 and C2) born to a G4P2 group O mother and A2B father were positive for blood group B, and had neonatal thrombocytopenia (TP) (C1 = 33K/μL, C2 = 61K/μL), anemia, positive direct antiglobulin test, elevated reticuloytes and hyperbilirubinemia requiring phototherapy. C1 required 3 platelet transfusions and RBC transfusion, and C2 required RBC transfusion. Both recovered in the immediate neonatal period. A third child (C3) inherited blood group A2 from the father and was born with a normal platelet count. The parents were incompatible for HPA-2b and -3b, but no platelet-specific antibodies were detected in maternal serum. High titer IgG antibodies were detected in maternal serum against father's platelets in both flow cytometry and modified antigen capture ELISA. This activity was completely removed by absorption with normal, washed group B RBCs. When tested by flow cytometry with monoclonal anti-B and anti-A, the father's platelets were shown to carry 17 times the normal level of B antigen, and only trace amounts of A antigen. We previously showed that the potent glycosyltransferase activity associated with the H-Exp trait causes essentially all H antigen on platelets and RBCs to be converted to A and/or B antigen. Consistent with this, father's platelets and RBCs were found to express no detectable H. Quantitation of B and H antigens on platelets and RBCs from C1 and C2 is pending receipt of samples. Findings made in this family indicate that maternal anti-B (and presumably anti-A) IgG antibodies can cause NATP in infants with the ABO “high expresser” trait. Maternal-fetal ABO incompatibility should be considered as a cause of NATP when maternal antibodies against platelet-specific antigens cannot be demonstrated. The possibility that ABO incompatibility can aggravate thrombocytopenia caused by antibodies against recognized platelet-specific antigens also deserves consideration.

Cost‐effectiveness of pathogen inactivation for platelet transfusions in the Netherlands

The objective of this study is to estimate cost-effectiveness of pathogen inactivation for platelet transfusions in the Netherlands. We used decision tree analysis to evaluate the cost-effectiveness of the addition of pathogen inactivation of pooled platelets to standard procedures for platelet transfusion safety (such as, donor recruitment and screening). Data on transfusions were derived from the University Medical Centre Groningen (the Netherlands) for 1997. Characteristics of platelet recipients (patient group, age, gender and survival) and data/assumptions on viral and bacterial risks were linked to direct and indirect costs/benefits of pathogen inactivation. Post-transfusion survival was simulated with a Markov model. Standard methods for cost-effectiveness were used. Cost-effectiveness was expressed in net costs per life-year gained (LYG) and estimated in baseline- and sensitivity analysis. Sensitivity was analysed with respect to various assumptions including sepsis risk, reduction of the discard rate and discounting. Stochastic analysis to derive 90% simulation intervals (SIs) was performed on sepsis risk. Net costs per LYG for pathogen inactivation were estimated 554,000 euro in the baseline-weighted average over the three patient groups (90% SI: 354,000-1092,500 euro). Sensitivity analysis revealed that cost-effectiveness was insensitive to viral risks and indirect costing, but highly sensitive to the assumed excess transfusions required and discounting of LYG. Given relatively high net costs per LYG that are internationally accepted for blood transfusion safety interventions, our estimated cost-effectiveness figures for pathogen inactivation may reflect acceptable cost-effectiveness in this specific area. Two main assumptions of our model were that the pathogen inactivation was 100% effective in preventing transmission of the pathogens considered and was not associated with major and/or costly adverse reactions. Validation of several crucial parameters is required, in particular the Dutch risk for acquiring and dying of transfusion-related sepsis.

Assessment of the correlation of platelet morphology with in vivo recovery and survival

There is continuing interest in the development of in vitro tests evaluating the in vivo function, recovery, and survival of platelets stored for transfusion. A recent forum concluded that no completely reliable test exists, although discoid morphology indicates a platelet's good health. We evaluated a novel device, the NAPSAC (Noninvasive Assessment of Platelet Shape and Concentration), designed to determine noninvasively the proportion of discoid platelets in a stored concentrate, as well as platelet concentration. Twenty-eight plateletapheresis concentrates stored 24 hours in PL-146 were evaluated. Percent discoid platelet results were correlated with radiolabeled autologous recovery and survival performed using 111Indium oxyquinoline and calculated using linear (L) and multiple-hit (M) models. pH of 8 concentrates was raised at the end of storage with 6N NaOH. Platelet concentration measured by NAPSAC and Coulter Thrombocounter C was compared in 256 plateletapheresis products. Percent discoid platelets at 24 hours did not correlate significantly with platelet recovery or survival (recovery L = 0.29, M = 0.28; survival L = 0.16, M = 0.03). Raising the pH (mean 6.38 to 6.94) resulted in a significant increase in percent discoid platelets (21% to 41%). Platelet concentration values for both methods studied were linearly correlated with a slope of 1.01 +/- 0.03, r = 0.81. Percent discoid platelets was not predictive of posttransfusion platelet recovery or survival. The results suggest that non-discoid platelets may survive posttransfusion and even revert to discoid shape, since raising the pH approximately doubled the percent of discoid platelets. The NAPSAC was shown to be a reliable instrument for noninvasively determining platelet concentration in PL-146 concentrates.