Urinary di-(2-ethylhexyl) phthalate metabolites for detecting transfusion of autologous blood stored in plasticizer-free bags

BACKGROUND : Autologous blood transfusion (ABT) efficiently increases sport performance and is the most challenging doping method to detect. Current methods for detecting this practice center on the plasticizer di(2-ethlyhexyl) phthalate (DEHP), which enters the stored blood from blood bags. Quantification of this plasticizer and its metabolites in urine can detect the transfusion of autologous blood stored in these bags. However, DEHP-free blood bags are available on the market, including n-butyryl-tri-(n-hexyl)-citrate (BTHC) blood bags. Athletes may shift to using such bags to avoid the detection of urinary DEHP metabolites.
STUDY DESIGN AND METHODS : A clinical randomized double-blinded two-phase study was conducted of healthy male volunteers who underwent ABT using DEHP-containing or BTHC blood bags. All subjects received a saline injection for the control phase and a blood donation followed by ABT 36 days later. Kinetic excretion of five urinary DEHP metabolites was quantified with liquid chromatography coupled with tandem mass spectrometry.
RESULTS : Surprisingly, considerable levels of urinary DEHP metabolites were observed up to 1 day after blood transfusion with BTHC blood bags. The long-term metabolites mono-(2-ethyl-5-carboxypentyl) phthalate and mono-(2-carboxymethylhexyl) phthalate were the most sensitive biomarkers to detect ABT with BTHC blood bags. Levels of DEHP were high in BTHC bags (6.6%), the tubing in the transfusion kit (25.2%), and the white blood cell filter (22.3%).
CONCLUSIONS : The BTHC bag contained DEHP, despite being labeled DEHP-free. Urinary DEHP metabolite measurement is a cost-effective way to detect ABT in the antidoping field even when BTHC bags are used for blood storage (read more).

Efficacy of transfusion with granulocytes from G-CSF/dexamethasone–treated donors in neutropenic patients with infection

High-dose granulocyte transfusion therapy has been available for 20 years, yet its clinical efficacy has never been conclusively demonstrated. We report here the results of RING (Resolving Infection in Neutropenia with Granulocytes), a multicenter randomized controlled trial designed to address this question. Eligible subjects were those with neutropenia (absolute neutrophil count <500/μL) and proven/probable/presumed infection. Subjects were randomized to receive either (1) standard antimicrobial therapy or (2) standard antimicrobial therapy plus daily granulocyte transfusions from donors stimulated with granulocyte colony-stimulating factor (G-CSF) and dexamethasone. The primary end point was a composite of survival plus microbial response, at 42 days after randomization. Microbial response was determined by a blinded adjudication panel. Fifty-six subjects were randomized to the granulocyte arm and 58 to the control arm. Transfused subjects received a median of 5 transfusions. Mean transfusion dose was 54.9 × 109 granulocytes. Overall success rates were 42% and 43% for the granulocyte and control groups, respectively (P > .99), and 49% and 41%, respectively, for subjects who received their assigned treatments (P = .64). Success rates for granulocyte and control arms did not differ within any infection type. In a post hoc analysis, subjects who received an average dose per transfusion of ≥0.6 × 109 granulocytes per kilogram tended to have better outcomes than those receiving a lower dose. In conclusion, there was no overall effect of granulocyte transfusion on the primary outcome, but because enrollment was half that planned, power to detect a true beneficial effect was low (read more)

No evidence of transmission of chronic lymphocytic leukemia through blood transfusion

Monoclonal B-cell lymphocytosis (MBL) is a precursor of chronic lymphocytic leukemia (CLL). Observations of MBL in blood donors raise concern that transmitted MBL may cause recipient CLL. Using a database with health information on 1.5 million donors and 2.1 million recipients, we compared CLL occurrence among 7413 recipients of blood from 796 donors diagnosed with CLL after donation cessation, and among 80 431 recipients of blood from 7477 matched CLL-free donors. During follow-up, 12 and 107 cases of CLL occurred among the exposed and unexposed recipients, respectively, yielding a relative risk of 0.94 (95% confidence interval, 0.52-1.71). Analyses using the entire database showed no evidence of CLL clustering among recipients of blood from individual donors. In conclusion, when donor MBL was approximated by subsequent donor CLL diagnosis, data from 2 countries’ entire computerized transfusion experience over more than 30 years indicate that MBL/CLL transmission does not contribute importantly to recipient CLL risk (read more)

Exposure to ABO-nonidentical blood associated with increased in-hospital mortality in patients with group A blood

BACKGROUND : Transfusing ABO-compatible blood avoids most acute hemolytic reactions, but donor units that are ABO compatible are not necessarily ABO identical. Emerging data have raised concerns that ABO-nonidentical blood products lead to adverse outcomes.
STUDY DESIGN AND METHODS : A large multihospital registry (Transfusion Registry for Utilization, Surveillance, and Tracking) was used to determine the association between exposure to ABO-nonidentical blood and in-hospital mortality. Cox regression analyses controlled for sex, age, hemoglobin, creatinine, and in-hospital interventions and stratified by age of blood and admission year.
RESULTS : Data from 18,843 non–group O patients admitted between 2002 and 2011 and receiving at least 1 unit of blood were analyzed. Overall, group A patients had significantly increased risk of in-hospital death upon receiving a nonidentical unit (RR , 1.79; 95% CI, 1.20-2.67; p = 0.005). There was no evidence of increased risk for group B or AB patients. Similar results were seen when only patients with circulatory disorders were considered. When patients with injury or poisoning were excluded, both group A and B patients had significantly increased risk of in-hospital death.
CONCLUSION : Our study demonstrates an adverse effect of ABO-nonidentical blood in a broad range of patients with group A blood, after adjustment for potential confounders. Further research in this area is required to study possible mechanisms. Increased mortality associated with exposure to nonidentical blood in these patients would have a substantial impact at the population level; it would challenge how blood suppliers manage inventory and recruit donors and how health care providers administer blood (read more and editorial).

Variable HLA expression on deceased donor lymphocytes: Not all crossmatches are created equal

Flow cytometric crossmatch tests are used to detect donor-specific antibody and determine eligibility for transplantation. Crossmatch sensitivity is dependent on lymphocyte quality, to include HLA expression on the cell surface. The impact of HLA expression variability on crossmatch reactivity was examined using lymphocytes isolated from different donor types: deceased donor (DD) versus living donor (LD) and tissue sources (blood, spleen, or lymph nodes). HLA class I expression was similar on B cells isolated from LD blood, DD spleen, and DD lymph nodes, but significantly lower on B cells isolated from DD blood (p=0.0004). In contrast, class II expression on B cells and class I on T cells were significantly higher in LD blood than all DD tissues. Within DD tissues, spleen provided the highest expression of class II on B cells and class I on T cells. HLA expression on B cells, but not T cells, was impacted by memory (CD27+) versus non-memory status. Importantly, HLA expression differences on lymphocytes isolated from the same donor but different tissues impacted crossmatch outcomes. HLA expression is impacted by multiple factors and should be routinely monitored to ensure crossmatch sensitivity and to reconcile crossmatch strength with solid phase HLA antibody analyses (read more)

Survival after ultramassive transfusion: a review of 1360 cases

BACKGROUND : Information about patient survival after transfusion of multiple blood volumes is limited, and most reports have focused on trauma patients.
STUDY DESIGN AND METHODS : Retrospective study of blood use and survival at 11 hospitals in six nations between 2009 and 2013. Ultramassive transfusion (UMT) was defined as transfusion of 20 or more red blood cell (RBC) units over the course of any 2 consecutive calendar days.
RESULTS : A total of 1975 patients received UMT and a representative sample of 1360 patients was studied in detail. Patients were grouped into seven diagnostic categories: solid organ transplantation (n = 411), cardiac or major vascular surgery (n = 317), general surgery (n = 228), trauma (n = 221), general medicine (n = 124), obstetrics (n = 23), and other (n = 36). During the 7 days after initiation of UMT, these patients used more than 120,000 blood components. The median (interquartile range) blood use was 35 (26-50) RBC units, 30 (20-47) plasma units, and 7 (4-13) platelet doses. Five- and 30-day survival significantly declined with increasing RBC use. Overall survivals of patients receiving UMT were 71% (5 day) and 60% (30 day), and in the subset of 165 patients receiving 60 or more RBC units over 2 consecutive days, 5-day survival was 54% ranging from 17% (trauma) to 75% (solid organ transplant). The decline in survival with increasing RBC transfusions was minimal for patients undergoing solid organ transplantation and was most pronounced for trauma and nonsurgical bleeding patients.
CONCLUSION : Trauma was not the leading cause of UMT. Increasing RBC requirements were significantly associated with decreasing survival. However, survival was more strongly associated with diagnostic category than total RBCs transfused, with highest survival rates in solid organ transplant surgery (read more).

Platelets made HLA deficient by acid treatment aggregate normally and escape destruction by complement and phagocytes in the presence of HLA antibodies

BACKGROUND : The presence of antibodies against HLA Class I can lead to platelet (PLT) transfusion refractoriness, that is, the repeated failure to achieve adequate posttransfusion PLT count increments. PLT refractoriness can be overcome by transfusion of HLA-matched donor PLTs. A different approach is to remove HLA from the PLT surface using low pH. Previous case studies using HLA-stripped PLTs showed encouraging but inconsistent results and lacked information on the biologic effects of acid treatment on PLT function as well as sensitivity to PLT destruction in the presence of HLA antibodies.
STUDY DESIGN AND METHODS : PLTs prepared from buffy coats were stripped from HLA Class I using a brief incubation at pH 2.9. Kinetics of acid stripping, viability, phenotypic alterations, and sensitivity to complement-mediated lysis and phagocytosis were determined by flow cytometry. Functional potential was evaluated using a multiplate analyzer.
RESULTS : Acid-treated PLTs were viable, upregulated activation markers normally and aggregated to a similar extent as untreated PLTs in response to stimulation with three natural agonists. Acid treatment removed 70% to 90% of HLA Class I complexes from the PLT surface, which led to complete protection from HLA antibody–mediated complement lysis and reduced monocyte-mediated phagocytosis in the presence of anti-HLA in vitro.
CONCLUSION : Our study fills an important knowledge gap in how acid treatment affects PLT function and interactions with immune cells, paving the way for controlled clinical trials to evaluate acid-treated PLTs as an alternative to HLA-matched donors in PLT refractoriness (read more)

Five-day stability of thawed plasma: solvent/detergent-treated plasma comparable with fresh-frozen plasma and plasma frozen within 24 hours

BACKGROUND : Plasma stored refrigerated for up to 5 days after thawing is common practice in many US hospitals. Therefore, clotting factor activities in fresh-frozen plasma (FFP), plasma frozen within 24 hours (PF24), and solvent/detergent-treated plasma (SDP), thawed and stored at 1 to 6°C for up to 5 days, were investigated.
STUDY DESIGN AND METHODS : Five A, B, O, and AB units of FFP, PF24, and SDP were thawed and maintained for 5 days at 1 to 6°C. The activity of factor (F)V, FVII, FVIII, protein S (PS), and ADAMTS13 was determined in each unit at baseline and every 24 hours thereafter for 5 days.
RESULTS : After thaw, mean values of the variables tested were within the normal range in all three plasma products although, in SDP, FVIII activity was significantly lower (p = 0.0039). After 5 days of storage all factors significantly declined except for ADAMTS13 activity, which was stable. Mean FVIII and ADAMTS13 activity was comparable in all three plasma products and within the normal range, mean FV activity was significantly lower in FFP and PF24 (p<0.0001) compared to SDP, and mean FVII activity was significantly lower in PF24 (p<0.03) than in FFP or SDP. Mean PS activity was below the normal range in all three plasma products with the lowest values in SDP (p = 0.0001).
CONCLUSION : Over 5 days of refrigerated storage the changes in the measured coagulation factors in FFP, PF24, and SDP are comparable. Clinical follow-up is needed to assess whether slightly lower PS levels in SDP are clinically important (read more)

Automated cold temperature cycling improves in vitro platelet properties and in vivo recovery in a mouse model compared to continuous cold storage

BACKGROUND : Platelets (PLTs) stored at cold temperatures (CTs) for prolonged time have dramatically reduced bacterial growth but poor survival when infused. A previous study demonstrated that human PLTs stored with manual cycling between 4°C (12 hr) and 37°C (30 min) and infused into severe combined immunodeficient (SCID) mice had survivals similar to or greater than those stored at room temperature (RT). In this study, the in vitro and in vivo properties of PLTs stored in an automated incubator programmed to cycle between 5°C (11 hr) and 37°C (1 hr) were evaluated.
STUDY DESIGN AND METHODS : A Trima apheresis unit (n = 12) was aliquoted (60 mL) in CLX bags. One sample was stored with continuous agitation (RT), a second sample was stored at 4-6°C without agitation (CT), and a third sample was placed in an automated temperature cycler with 5 minutes of agitation during the warm-up period (thermocycling [TC]). PLTs were assayed for several relevant quality variables. On Day 7, PLTs were infused into SCID mice and in vivo recovery was assessed at predetermined time points after transfusion.
RESULTS : The glucose consumption rate, morphology score, hypotonic shock recovery level, and aggregation levels were increased and mitochondrial reactive oxygen species accumulations were decreased in TC-PLTs compared to those of CT-PLTs. The pH and Annexin V binding were comparable to those of RT-PLTs. All TC-PLTs had greater recovery than CT-PLTs and were comparable to RT-PLTs.
CONCLUSION : PLTs stored under automated TC conditions have improved in vivo recovery and improved results for a number of in vitro measures compared to CT-PLTs (read more)

Assisted reproductive technology: an uncommon, but increasing, cause of parent-child ABO discrepancy

In the course of evaluating an infant with mild ABO hemolytic disease of the fetus or newborn, we detected an unexpected discrepancy between the mother’s group O phenotype and the infant’s group AB phenotype. While we were considering the possibilities of a blood sample or patient misidentification, cis-AB or Bombay phenotype, we learned that the mother had attended a fertility clinic and her pregnancy was induced using a donor egg (oocyte). The newborn’s total bilirubin peaked at 5 mg/dL and returned to a normal concentra-tion with phototherapy. Mother and child were dis-charged uneventfully 48 hours after the delivery (read more)

Specifications for anti-A and anti-B in intravenous immunoglobulin: history and rationale

Intravenous immunoglobulin (IVIG) products are generally safe and efficacious, although treatment-related adverse reactions can occur in recipients. Adverse reactions include hemolysis in non–blood group O recipients linked to the passive transfer of anti-A and/or anti-B present in the fractionated immunoglobulin product. In light of the recent increase in reported cases of severe hemolysis associated with anti-A and/or anti-B, this article traces the development of pharmacopoeial specifications, tests, and reference reagents to control their titers in IVIG products (read more)

A population-based study on blood lead levels in blood donors

BACKGROUND : Recent studies suggested that blood transfusion may represent a significant source of lead exposure in premature infants. Objectives of this study were to determine blood lead levels (BLLs) in a representative sample of blood donors and to identify risk factors associated with BLLs of 0.15 µmol/L or more.
STUDY DESIGN AND METHODS : A study was conducted in 2006 to 2007 in 49 drive sites in Quebec. Individuals who qualified for blood donation were eligible to participate. Information was harvested from blood donor file and a standardized self-administered questionnaire. Lead analysis was performed by inductively coupled plasma mass spectrometry. Data on Quebec blood donors from 2003 to 2006 (n = 320,543) were used to establish a reference population. Geometric mean (GM) and 95% confidence interval (CI) were used to describe the results. The project was approved by an ethics committee.
RESULTS : Of 6715 eligible individuals, 3490 participated (1392 women and 2098 men). Their mean age was 46.5 years. Results were weighted for region, sex, and age. The GM of BLLs was 0.082 µmol/L (95% CI, 0.027-0.247; range, 0.011-2.90 µmol/L). BLLs of more than 0.15 µmol/L were found in 15.5% of participants. In multivariate analysis, BLLs were mainly explained by age and sex of participants (p < 0.001). A significant association was also found between BLLs and the region of residence, education level, dwelling age, occupational and leisure activities at high risk for lead exposure, smoking, and alcohol intake (p < 0.001).
CONCLUSION : BLL in blood donors is strongly explained by sex and age, a fact that can be taken into consideration when transfusing neonates (read more)

Implementing mass-scale red cell genotyping at a blood center

BACKGROUND : When problems with compatibility beyond ABO and D arise, currently transfusion services search their inventories and perform time-consuming serologic testing to locate antigen-negative blood. These clinically important blood group antigens can be detected reliably by red cell genotyping, which is a technology whereby DNA-based techniques are used to evaluate gene polymorphisms that determine the expression of blood group antigens. We introduced mass-scale genotyping and measured availability of genotyped blood.
STUDY DESIGN AND METHODS : All non-Caucasian donors qualified for genotyping along with donors who had a history of repeat donation. Mass-scale red cell genotyping, performed on an electronic interfaced open array platform, was implemented to screen blood donors for 32 single-nucleotide polymorphisms that predicted 42 blood group antigens. Genotype screening results were confirmed by phenotyping, when needed for antigen-negative transfusion, before release of the red blood cell (RBC) unit.
RESULTS : Approximately 22,000 donors were red cell genotyped within 4 months and a total of 43,066 donors in 4 years. There were 463 discordances (0.52% of 89,596 genotypes with a phenotype). Among the 307 resolved discordances, approximate equal numbers represented historical serologic or genotyping discrepancies (n = 151 and n = 156, respectively). In the final year of the study, a mean of 29% of the daily inventory had a genotype.
CONCLUSIONS : Red cell genotyping of blood donors using an electronic interface created a large and stable supply of RBC units with historical genotypes. The database served the needs of antigen-negative blood requests for a large regional blood center and allowed us to abandon screening by serology (read more)

Cost implications of implementation of pathogen-inactivated platelets

BACKGROUND : Pathogen inactivation (PI) is a new approach to blood safety that may introduce additional costs. This study identifies costs that could be eliminated, thereby mitigating the financial impact.
STUDY DESIGN AND METHODS : Cost information was obtained from five institutions on tests and procedures (e.g., irradiation) currently performed, that could be eliminated. The impact of increased platelet (PLT) availability due to fewer testing losses, earlier entry into inventory, and fewer outdates with a 7-day shelf life were also estimated. Additional estimates include costs associated with managing 1) special requests and 2) test results, 3) quality control and proficiency testing, 4) equipment acquisition and maintenance, 5) replacement of units lost to positive tests, 6) seasonal or geographic testing, and 7) health department interactions.
RESULTS : All costs are mean values per apheresis PLT unit in USD ($/unit). The estimated test costs that could be eliminated are $71.76/unit and a decrease in transfusion reactions corresponds to $2.70/unit. Avoiding new tests (e.g., Babesia and dengue) amounts to $41.80/unit. Elimination of irradiation saves $8.50/unit, while decreased outdating with 7-day storage can be amortized to $16.89/unit. Total potential costs saved with PI is $141.65/unit. Costs are influenced by a variety of factors specific to institutions such as testing practices and the location in which such costs are incurred and careful analysis should be performed. Additional benefits, not quantified, include retention of some currently deferred donors and scheduling flexibility due to 7-day storage.
CONCLUSIONS : While PI implementation will result in additional costs, there are also potential offsetting cost reductions, especially after 7-day storage licensing (read more)

Routine filtration of hematopoietic stem cell products: the time has arrived

BACKGROUND : Most blood products are infused at the time of transfusion through a standard blood filter, designed to capture macroaggregates and cellular debris that might be harmful to the patient if infused. Hematopoietic stem cell products are not universally filtered, likely due to concern about loss of viable stem cells in the filtration process.
STUDY DESIGN AND METHODS : We conducted a two-phase study to better understand the safety of routine filtration. First, surplus cryopreserved stem cell products were thawed and filtered, with markers of viability and potency measured. Second, routine filtration was implemented as part of routine practice at our center, and date of neutrophil and platelet (PLT) recovery was compared to historical controls.
RESULTS : In the first phase, there was no difference seen in any markers of viability or potency for products after routine filtration. Based on those results, routine filtration was implemented. There was no difference in neutrophil or PLT engraftment. Thus, in this study, routine filtration did not impact the number of viable stem cells and did not delay engraftment.
CONCLUSION : Given the very real harm posed by infusion of macroaggregates and cellular debris, and no clear disadvantage to filtration, routine filtration of stem cell products should be considered the standard of care (read more).

When blood transfusion medicine becomes complicated due to interference by monoclonal antibody therapy

BACKGROUND : Monoclonal antibodies (MoAbs) are increasingly integrated in the standard of care. The notion that therapeutic MoAbs can interfere with clinical laboratory tests is an emerging concern that requires immediate recognition and the development of appropriate solutions. Here, we describe that treatment of multiple myeloma patients with daratumumab, a novel anti-CD38 MoAb, resulted in false-positive indirect antiglobulin tests (IATs) for all patients for 2 to 6 months after infusion. This precluded the correct identification of irregular blood group antibodies for patients requiring blood transfusion.
STUDY DESIGN AND METHODS : The IAT was performed using three- and 11-donor-cell panels. Interference of daratumumab and three other anti-CD38 MoAbs was studied using fresh-frozen plasma spiked with different MoAb concentrations. Additionally it was tested whether two potentially neutralizing agents, anti-idiotype antibody and recombinant soluble CD38 (sCD38) extracellular domain, were able to inhibit the interference.
RESULTS : The CD38 MoAbs caused agglutination in the IAT in a dose-dependent manner. Addition of an excess of anti-idiotype antibodies or sCD38 protein to the test abrogated CD38 MoAb interference and successfully restored irregular antibody screening and identification.
DISCUSSION : CD38 MoAb therapy causes false-positive results in the IAT. The reliability of the test could be restored by adding a neutralizing agent against the CD38 MoAb to the patient's plasma. This study emphasizes that during drug development, targeted therapeutics should be investigated for potential interference with laboratory tests. Clinical laboratories should be informed when patients receive MoAb treatments and matched laboratory tests to prevent interference should be employed (read more)

Anti-A and anti-B titers in donor plasma, plasma pools, and immunoglobulin final products

BACKGROUND : High-dose intravenous immunoglobulin (IVIG) treatments are implicated in hemolytic events in some patients receiving treatment. The passive transfer of IgG anti-A and anti-B agglutinin is thought to play a role in the development of these events. The purpose of this study was to determine the prevalence of high-titer IgG anti-A and anti-B in plasma donors and investigate if there is any advantage of excluding these donors from the donor pool to limit anti-A and anti-B content in IVIG product.
STUDY DESIGN AND METHODS : IgG anti-A and anti-B levels were assessed from group O donor plasma, manufacturing IgG plasma pools, and finished IVIG product (Gammagard Liquid). Antibody level in group O donors was also assessed by sex and age for their relative contribution of antibody to the plasma pool.
RESULTS : The majority of group O donors (80%) had antibody titers of less than 1000. Of those with titers of at least 1000, theoretical estimates provide further evidence that the effects of high-titer donors are minimal. Antibody levels in plasma pools both during the manufacturing process and from the final IVIG product also support that anti-A and anti-B levels are low. In general, there were more females than males with higher antibody titer levels, with significantly more females than males with anti-A.
CONCLUSION : Excluding donors with high anti-A and anti-B titers has minimal impact on the finished IVIG product titers due to ABO antibody neutralization and the dilution factor in the manufacturing pool (read more).