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BACKGROUND: In hematologic and transfusion medicine research, measurement of red blood cell (RBC) in vivo kinetics must be safe and accurate. Recent reports indicate use of biotin-labeled RBC (BioRBC) to determine red cell survival (RCS) offers substantial advantages over (51)Cr and other labeling methods. Occasional induction of BioRBC antibodies has been reported. STUDY DESIGN AND METHODS: To investigate the causes and consequences of BioRBC immunization, we reexposed three previously immunized adults to BioRBC and evaluated the safety, antibody emergence, and RCS of BioRBC. RESULTS: BioRBC re-exposure caused an anamnestic increase of plasma BioRBC antibodies at 5–7 days; all were subclass IgG(1) and neutralized by biotinylated albumin, thus indicating structural specificity for the biotin epitope. Concurrently, specific antibody binding to BioRBC was observed in each subject. As biotin label density increased, the proportion of BioRBC that bound increased antibody also increased; the latter was associated with proportional accelerated removal of BioRBC labeled at density 6 μg/mL. In contrast, only one of three subjects exhibited accelerated removal of BioRBC density 2 μg/mL. No adverse clinical or laboratory events were observed. Among three control subjects who did not develop BioRBC antibodies following initial BioRBC exposure, re-exposure induced neither antibody emergence nor accelerated BioRBC removal. DISCUSSION: We conclude re-exposure of immunized subjects to BioRBC can induce anamnestic antibody response that can cause an underestimation of RCS. To minimize chances of antibody induction and underestimation of RCS, we recommend an initial BioRBC exposure volume of ≤10 mL and label densities of ≤18 μg/mL.

Author(s): Gottschall, Jerome; Wu, YanYun; Triulzi, Darrell; Kleinman, Steven; Strauss, Ronald; Zimrin, Ann Butler; McClure, Christopher; Tan, Sylvia; Bialkowski, Walter; Murphy, Edward; Ness, Paul; NHLBI Recipient Epidemiology and Donor Evaluation (REDS-III) Study | Abstract: BackgroundUsing the Recipient and Donor Epidemiology Study-III (REDS-III) recipient and donor databases, we performed a retrospective analysis of platelet use in 12 US hospitals that were participants in REDS-III.Study design and methodsData were electronically extracted from participating transfusion service and blood center computer systems and from medical records of the 12 REDS-III hospitals. All platelet transfusions from 2013 to 2016 given to patients aged 18 years and older were included in the analysis.ResultsThere were 28,843 inpatients and 2987 outpatients who were transfused with 163,719 platelet products (103,371 apheresis, 60,348 whole blood derived); 93.5% of platelets were leukoreduced and 72.5% were irradiated. Forty-six percent were transfused to patients with an International Classification of Diseases, 9th/10th Revision (ICD-9/10) diagnosis of leukemia, myelodysplastic syndrome (MDS), or lymphoma. The general ward and the intensive care unit (ICU) were the most common issue locations. Only 54% of platelet transfusions were ABO identical; and 60.6% of platelet transfusions given to Rh-negative patients were Rh positive. The most common pretransfusion platelet count range for inpatients was 20,000 to 50,000/μL, for outpatients it was 10,000 to 20,000/μL. Among ICU patients, 35% of platelet transfusion episodes had a platelet count of greater than 50,000/μL; this was only 8% for general ward and 2% for outpatients. The median posttransfusion increment, not corrected for platelet dose and/or patient size, ranged from 12,000 to 20,000/μL for inpatients, and from 17,000 to 27,000/μL for outpatients.ConclusionsThese data from one of the largest reviews of platelet transfusion practice to date provide guidance for where to focus future clinical research studies and platelet blood management programs.

The current reference method in the United States for measuring in vivo population red blood cell (RBC) kinetics utilizes chromium-51 (51 Cr) RBC labeling for determining RBC volume, 24-hour posttransfusion RBC recovery, and long-term RBC survival. Here we provide evidence supporting adoption of a method for kinetics that uses the biotin-labeled RBCs (BioRBCs) as a superior, versatile method for both regulatory and investigational purposes. RBC kinetic analysis using BioRBCs has important methodologic, analytical, and safety advantages over 51 Cr-labeled RBCs. We critically review recent advances in labeling human RBCs at multiple and progressively lower biotin label densities for concurrent, accurate, and sensitive determination of both autologous and allogeneic RBC population kinetics. BioRBC methods valid for RBC kinetic studies, including successful variations used by the authors, are presented along with pharmacokinetic modeling approaches for the accurate determination of RBC pharmacokinetic variables in health and disease. The advantages and limitations of the BioRBC method-including its capability of determining multiple BioRBC densities simultaneously in the same individual throughout the entire RBC life span-are presented and compared with the 51 Cr method. Finally, potential applications and limitations of kinetic BioRBC determinations are discussed.

Background Although the subject of many previous studies, the importance of white blood cell (WBC) alloimmunization in granulocyte transfusion therapy has not been settled. In this study, we report the results of the effects of WBC antibodies in the RING (Resolving Infection in Neutropenia with Granulocytes) study, a randomized controlled trial comparing the efficacy of daily granulocyte transfusion therapy plus antimicrobials versus antimicrobials alone; the primary outcome results have been published previously. Study design and methods One hundred fourteen subjects were enrolled in the study. Serum samples for WBC antibody determination were obtained from each subject at baseline and at 2 and 6 weeks. One hundred subjects had at least one antibody test result. Samples were tested for human leukocyte antigen (HLA) Class I and Class II antibodies as well as for granulocyte-specific antibodies using granulocyte agglutination and immunofluorescence techniques. All testing was performed at a central laboratory. Results Baseline WBC alloimmunization was modest, depending somewhat on the assay. Seroconversion during the study was slightly higher in the granulocyte transfusion arm, but the differences were not statistically significant. There was no demonstrable effect of the presence of alloimmunization on the primary outcome (survival and microbial response at 42 days), the occurrence of transfusion reactions (either overall or pulmonary), or posttransfusion neutrophil increments. Conclusion The presence or development of WBC antibodies had no demonstrable effect on any clinical aspect of granulocyte transfusion therapy. It appears that, at least in the patient population studied, there is no evidence suggesting need for concern about recipient WBC alloimmunization when prescribing granulocyte transfusions.

BACKGROUND Critically ill preterm very-low-birthweight (VLBW) neonates (birthweight ≤ 1.5 kg) frequently develop anemia that is treated with red blood cell (RBC) transfusions. Although RBCs transfused to adults demonstrate progressive decreases in posttransfusion 24-hour RBC recovery (PTR24 ) during storage-to a mean of approximately 85% of the Food and Drug Administration-allowed 42-day storage-limited data in infants indicate no decrease in PTR24 with storage. STUDY DESIGN AND METHODS We hypothesized that PTR24 of allogeneic RBCs transfused to anemic VLBW newborns: 1) will be greater than PTR24 of autologous RBCs transfused into healthy adults and 2) will not decrease with increasing storage duration. RBCs were stored at 4°C for not more than 42 days in AS-3 or AS-5. PTR24 was determined in 46 VLBW neonates using biotin-labeled RBCs and in 76 healthy adults using 51 Cr-labeled RBCs. Linear mixed-model analysis was used to estimate slopes and intercepts of PTR24 versus duration of RBC storage. RESULTS For VLBW newborns, the estimated slope of PTR24 versus storage did not decrease with the duration of storage (p = 0.18) while for adults it did (p

Background Biotin-labeled red blood cells (BioRBCs) are used for in vivo kinetic studies. Because BioRBC dosing occasionally induces antibodies, a sensitive and specific anti-BioRBC detection assay is needed. Study design and methods Aims were to 1) develop a gel card assay to evaluate existing, naturally occurring and BioRBC-induced plasma antibodies, 2) compare gel card and tube agglutination detection results, and 3) test for a relationship of antibody induction and BioRBC dose. Reagent BioRBCs were prepared using sulfo-NHS biotin ranging from densities 18 (BioRBC-18) to 1458 (BioRBC-1458) µg/mL RBCs. Results Among BioRBC-exposed subjects, gel card and tube agglutination results were concordant in 21 of 22 adults and all 19 infant plasma samples. Gel card antibody detection sensitivity was more than 10-fold greater than tube agglutination. Twelve to 16 weeks after BioRBC exposure, induced anti-antibodies were detected by gel card in three of 26 adults (12%) at reagent densities BioRBC-256 or less, but in none of 41 infants. Importantly, induced anti-BioRBC antibodies were associated with higher BioRBC dose (p = 0.008); no antibodies were detected in 18 subjects who received BioRBC doses less than or equal to BioRBC-18. For noninduced BioRBC antibodies, six of 1125 naive adults (0.3%) and none of 46 naive infants demonstrated existing anti-BioRBC antibodies using reagent BioRBC-140 or -162. Existing anti-BioRBCs were all neutralized by biotin compounds, while induced antibodies were not. Conclusions The gel card assay is more sensitive than the tube agglutination assay. We recommend reagent BioRBC-256 for identifying anti-BioRBCs. Use of a low total RBC biotin label dose (≤ BioRBC-18) may minimize antibody induction.

BACKGROUND Prevalence estimates of the serious hazards of transfusion vary widely. We hypothesized that the current reporting infrastructure in the United States fails to capture many transfusion reactions and undertook a multicenter study using active surveillance, data review, and adjudication to test this hypothesis. STUDY DESIGN AND METHODS A retrospective record review was completed for a random sample of 17% of all inpatient transfusion episodes over 6 months at four academic tertiary care hospitals, with an episode defined as all blood products released to a patient in 6 hours. Data were recorded by trained clinical research nurses, and serious reactions were adjudicated by a panel of transfusion medicine experts. RESULTS Of 4857 transfusion episodes investigated, 1.1% were associated with a serious reaction. Transfusion-associated circulatory overload was the most frequent serious reaction noted, being identified in 1% of transfusion episodes. Despite clinical notes describing a potential transfusion association in 59% of these cases, only 5.1% were reported to the transfusion service. Suspected transfusion-related acute lung injury/possible transfusion-related acute lung injury, anaphylactic, and hypotensive reactions were noted in 0.08, 0.02, and 0.02% of transfusion episodes, respectively. Minor reactions, including febrile nonhemolytic and allergic, were noted in 0.62 and 0.29% of transfusion episodes, respectively, with 30 and 50% reported to the transfusion service. CONCLUSION Underreporting of cardiopulmonary transfusion reactions is striking among academic, tertiary care hospitals. Complete and accurate reporting is essential to identify, define, establish pathogenesis, and mitigate/treat transfusion reactions. A better understanding of the failure to report may improve the accuracy of passive reporting systems.

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 count500/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 × 10(9) 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 × 10(9) 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. RING was registered at www.clinicaltrials.gov as #NCT00627393.

Background and objectives Many hospitals require transfusions to be discontinued when vital signs stray from predetermined ranges, regardless of clinical symptoms. Variations in vital signs may be unrelated to transfusion, however, and needlessly stopping a transfusion may delay medical care while increasing donor exposures and healthcare costs. We hypothesized that a detailed study of vital sign changes associated with transfusion of blood product by component, including those associated with potential reactions (complicated) and those deemed to be uncomplicated, would establish a useful framework of reference for treating clinicians and transfusion services alike. Materials and methods A retrospective electronic record review of transfusion service and transfusion recipient data was completed on 3852 inpatient transfusion episodes over a 6-month period at four academic tertiary care hospitals across the United States. Vital signs pre- and post-transfusion were recorded by trained clinical research nurses. Serious reactions were adjudicated by a panel of transfusion medicine experts. Results In both uncomplicated transfusions (n = 3765) and those including an adverse reaction (n = 87), vital sign fluctuations were generally modest. Compared to uncomplicated transfusions, transfusions complicated by febrile reactions were associated with higher pretransfusion temperature and higher pretransfusion pulse rates. Episodes of transfusion circulatory overload were associated with higher pretransfusion respiration rates compared to uncomplicated transfusions. Conclusion Most transfusions are associated with only modest changes in vital signs. Pretransfusion vital signs may be an important yet previously understudied predictor of vital sign changes during transfusion. The optimal role of vital sign assessment during blood transfusion deserves further study.

BACKGROUND How platelet (PLT) product characteristics such as dose, source (whole blood-derived (WBD) vs. apheresis), storage duration, and ABO matching status affect the risks of transfusion-related adverse events (TRAEs) is unclear. Similarly, more information is needed to define how recipient characteristics affect the frequency of TRAEs following PLT transfusion.

The purpose of this review is to report a recently completed multicenter randomized controlled trial of neutrophil/granulocyte transfusions collected from G-CSF + dexamethasone donors to treat neutropenic infections in oncology and transplant patients, within the context of other historic and current clinical trials.The multicenter trial (RING Study) was funded by the NHLBI transfusion medicine/hemostasis clinical trials network.There was no significant benefit of therapeutic neutrophil/granulocyte transfusions versus antibiotics per intention to treat analysis, but 32% of patients received substandard neutrophil doses. Separate analysis suggested patients given a higher neutrophil doses had better outcomes.Efficacy of 'high-dose' therapeutic neutrophil/granulocyte transfusions remains unproven, but promising.

S. Panzer, S. Engelbrecht, M. F. Cole-Sinclair, E. M. Wood, S. Wendel, S. Biagini, Z. Zhu, J.-J. Lefrere, G. Andreu, T. Zunino, J.-J. Cabaud, P. Rouger, O. Garraud, K. Janetzko, M. Muller-Steinhardt, P. van der Burg, A. Brand, P. Agarwal, T. Triyono, A. Gharehbaghian, N. Manny, O. Zelig, A. Takeshita, Y. Yonemura, H. Fujihara, K. E. Nollet, H. Ohto, K.-S. Han, V. S. Nadarajan, G. Berlin, S. G. Sandler, R. G. Strauss & H. W. Reesink

Background Prevention of red blood cell (RBC) alloimmunization in patients with sickle cell disease (SCD) focuses on phenotypic RBC matching. We assessed alloimmunization among transfused patients with SCD after implementing leukoreduction and prophylactic antigen matching (PAM). Study design and methods Retrospective review of transfusion and medical records for SCD patients 18 months to 81 years of age was performed covering two 5-year periods: Period 1, no PAM, occasional leukoreduction, and Period 2, consistent leukoreduction and extended PAM (Rh, Kell, S, Fy, Jk) for patients already alloimmunized. Patients transfused in Period 1 were excluded from Period 2. Results A total of 293 patients were transfused in Period 1 and 183 in Period 2. Median time between first sample and last type and screen after transfusion was 2.12 years in Period 1 and 1.03 years in Period 2. Initial alloimmunization prevalence was lower in Period 2 (26.2%) versus Period 1 (37.5%) and after subsequent transfusions in Period 2 (23.8%) versus Period 1 (45.7%), although without significant difference after adjusting for number of units transfused, percentage of leukoreduced RBCs, sex, and age. Alloimmunized patients received more nonleukoreduced RBCs in Period 1 than nonalloimmunized. Patients transfused during inflammatory conditions were not significantly more likely to become alloimmunized. Conclusions The prevalence of initial and subsequent RBC alloimmunization in Period 2 was lower than that in Period 1; however, overall prevalence remained high. We recommend leukoreduced, hemoglobin S-negative Rh and Kell PAM RBCs for transfusion of patients with SCD. Component and recipient factors affecting alloimmunization should be studied further.

Age-group analyses were conducted of patients in the prophylactic platelet dose trial (PLADO), which evaluated the relation between platelet dose per transfusion and bleeding. Hospitalized patients with treatment-induced hypoproliferative thrombocytopenia were randomly assigned to 1 of 3 platelet doses: 1.1 × 1011, 2.2 × 1011, or 4.4 × 1011 platelets/m2 per transfusion, given for morning counts of ≤ 10 000 platelets/μL. Daily hemostatic assessments were performed. The primary end point (percentage of patients who developed grade 2 or higher World Health Organization bleeding) was evaluated in 198 children (0-18 years) and 1044 adults. Although platelet dose did not predict bleeding for any age group, children overall had a significantly higher risk of grade 2 or higher bleeding than adults (86%, 88%, 77% vs 67% of patients aged 0-5 years, 6-12 years, 13-18 years, vs adults, respectively) and more days with grade 2 or higher bleeding (median, 3 days in each pediatric group vs 1 day in adults; P < .001). The effect of age on bleeding differed by disease treatment category and was most pronounced among autologous transplant recipients. Pediatric subjects were at higher risk of bleeding over a wide range of platelet counts, indicating that their excess bleeding risk may be because of factors other than platelet counts. This trial was registered at www.clinicaltrials.gov as #NCT00128713.

Summary Infections continue to be a serious problem for severely neutropenic oncology and haematopoietic progenitor cell (HPC) transplant patients. Although it is now possible to collect much larger numbers of neutrophils (PMNs) from donors stimulated with granulocyte colony-stimulating factor + corticosteroids, the efficacy of these ‘modern’ granulocyte/PMN transfusions, with higher doses of PMNs, has not been established by convincing randomized control trials. Accordingly, they cannot be recommended for standard therapy at this time.

Platelet characteristics, such as platelet dose, platelet source (apheresis vs pooled), platelet donor-recipient ABO compatibility, and duration of platelet storage, can affect posttransfusion platelet increments, but it is unclear whether these factors impact platelet transfusion efficacy on clinical bleeding. We performed secondary analyses of platelet transfusions given in the prospective randomized Platelet Dose Study, which included 1272 platelet-transfused hematology-oncology patients who received 6031 prophylactic platelet transfusions. The primary outcome of these analyses was time from first transfusion to first World Health Organization ≥ grade 2 bleeding. Platelet transfusion increments were assessed at 0.25 to 4 hours and 16 to 32 hours after platelet transfusion. There were 778 patients evaluable for analysis of time to bleeding. Adjusted models showed that randomized dose strategy, platelet source, ABO compatibility, and duration of storage did not predict this outcome. Platelet increments were generally higher for transfusions of apheresis platelets, ABO-identical platelets, and platelets stored 3 days versus 4 to 5 days. Thus, although platelet source, ABO compatibility, and duration of storage exert a modest impact on both absolute and corrected posttransfusion platelet increments, they have no measurable impact on prevention of clinical bleeding. This trial was registered at www.clinicaltrials.gov as #NCT00128713.

Characterization of the behavior of red blood cells (RBCs) in the bloodstream after transfusion (i.e., posttransfusion RBC kinetics) generally consists of measurement of RBC volume (RCV) and RBC survival (RCS) assessed by posttransfusion recovery at 24 hours (PTR24) and at later times, that is, to 50% survival (T50) and to the mean potential life span (MPL). Posttransfusion RBC kinetic studies provide information valuable in establishing the diagnosis and defining the pathogenesis of hematologic conditions and in determining optimal RBC transfusion and blood banking practices.1 Ideally, techniques for measuring posttransfusion RBC kinetics should be precise, safe, and easily applied. They should also be broadly applicable to diverse study populations and require minimal blood volumes for sampling, labeling, and analysis—particularly for infants. Methods utilizing chromium-51 (51Cr) radionuclide RBC labeling have been adopted by the International Committee for Standardization in Hematology as the reference method for determining posttransfusion RCS in humans.2 Unfortunately, because some species experience excessive rates of loss of 51Cr from RBCs (3%–4% per day in sheep, horses, cattle, goats, cats, and rats, compared to approx. 1% in humans),3 the 51Cr labeling method is not reliable for all mammalian species. Moreover, the costs for radiation containment and disposal are substantial and increasing. Recently, a method based on biotin-labeled RBCs (BioRBC) that does not require radiation exposure has been developed and validated for use in posttransfusion kinetic studies in a broad spectrum of mammalian species. In addition to overcoming the aforementioned limitations of the 51Cr method, the BioRBC method achieves similar sensitivity while requiring analytic blood volumes as small as 3 μL4,5 compared to the 1 to 2 mL recommended for the 51Cr method.3 The focus of this study was on validating the BioRBC labeling method in sheep. This is important because ovine fetal and newborn cardiovascular, pulmonary, and of relevance for this report, hematologic development and physiology resemble those of humans.6 In particular, ovine erythropoiesis in the fetus, neonate, and adult is similar to the human, and regulation of erythropoiesis and RBC kinetics in the ovine model are currently active areas of study.7–10 Accordingly, lambs and sheep are logical animal models for preclinical testing of this method for measuring RCV and RCS. We recently demonstrated that autologous sheep RBCs labeled at four discrete, low biotin densities yielded RCV results that are equivalent to one another and are within 10% of values determined simultaneously using [14C]cyanate-labeled RBCs.11 Further, we have applied the multidensity method to measurement of RCV in human adults with similar success. We were able to extend these human studies to survival of the BioRBCs and determine short-term and long-term RCS in eight participants. However, use of a radioactive-labeled marker such as 51Cr was not included because of the technical and financial demands. In this study, we extend these preclinical animal studies to assess the validity of RCS determined in the same animals using RBCs labeled with the same four biotin densities and with [14C]cyanate. Because of the additional complexities of dealing with an animal with a large spleen that sequesters up to 30% of RBCs, of the difficulties of measuring radiolabeled RBCs, and the need to fit a mathematical curve to accurately determine the proportion of each of the BioRBC peaks, we have only recently completed this analysis for the current report. To do so, we assessed short-term RCS by PTR24 and long-term RCS by T50 and by MPL. For long-term RCS, we hypothesized that these two variables would agree closely when determined using RBCs labeled with the lower BioRBC densities and that they would agree with RCS determined using [14C]cyanate.12

Red blood cells (RBCs) can be removed from the bloodstream at an accelerated rate by antibody-mediated mechanisms in a number of immunologic conditions including the following: 1) autoimmune hemolytic anemias, 2) transfusion of incompatible allogeneic RBCs into a recipient whose blood contains a corresponding alloantibody, 3) transfusion of passive antibodies into a recipient whose RBCs express the cognate antigen, 4) transplacental transfer of maternal allogeneic RBC antibodies into a fetus with RBCs expressing the cognate antigen resulting in alloimmune hemolytic disease of the fetus and newborn, and 5) posttransplantation transfer of viable donor lymphocytes capable of producing antibodies directed against recipient RBCs (e.g., group O donor into group A recipient). When caring for patients with these disorders, having an accurate and safe method for determining in vivo RBC kinetics would be of considerable value for establishing the diagnosis, defining the pathophysiology, and assessing the response to therapy. The availability of RBC survival (RCS) methods that address safety issues (e.g., avoidance of radioactivity) is particularly important for vulnerable patient populations including fetuses, neonates, children, and pregnant women. Direct assessment of RCS requires the ability to distinguish cohort-labeled RBCs from the remaining RBCs in the circulation and to express their number as the relative proportion of labeled RBCs to total RBCs in that blood sample. The internationally recognized reference method for measuring RCS uses autologous or allogeneic RBCs labeled with chromium 51 (51Cr) and is based on concentration of 51Cr per unit volume of blood.1 Because transfusion of 51Cr-labeled RBCs exposes the recipient to radiation, this approach is viewed as unacceptable for research studies in the human fetus, neonate, and pregnant woman. Our laboratory and others2-4 have developed methods for measuring RCS in humans based on labeling of RBCs with biotin at one5-10 or more11 densities. Here the term density refers to biotin labels per RBC and is determined by our selection of the concentration of biotinylating reagent per milliliter of RBCs in the biotinylation reaction mixture. Biotin-labeled RBCs (BioRBCs) are quickly and easily enumerated by flow cytometry.11 The biotin RBC method has the following safety and technical advantages over the standard 51Cr method: 1) study subjects including vulnerable populations are not exposed to radiation; 2) small blood volumes (e.g., 20 μL) are required for monitoring; and 3) multiple, independent RCS measurements can be made simultaneously in the same individual by labeling RBCs at more than one biotin density. The aim of this study was to determine whether RBCs labeled with biotin at a single density can be used to accurately measure shortened RCS of RBCs experimentally coated (opsonized) with antibody simulating the situation present in immune-mediated hemolytic anemia. We hypothesized that short-term and long-term RCS measured using BioRBCs would agree with RCS determined using RBCs labeled with 51Cr.

A previous International Forum (IF) addressed collection modalities of granulocytes (i.e. neutrophils) for supportive therapy (Vox Sang 2010; 88:567–575). Contributions to this IF came only from European centres because physicians representing centres from the United States of America (USA) elected not to participate. However, after publication of the IF, it was suggested by Professor Strauss that other centres from the USA be invited to describe their collection modalities, because it was anticipated that there might be noteworthy differences in practices between centres in Europe and the USA. Indeed, some differences have now become apparent, based on responses from four different centres in the USA. Question 1 Donor selection Do you collect granulocytes exclusively from your pool of healthy blood donors or do you also include the patients’ relatives as donors? When selecting the donor, do you predominantly select CMV-negative donors for CMV-negative recepients? Question 2 Mobilization regimen Does your national law allow the administration of G-CSF to unrelated donors Does your national law allow the administration of G-CSF to related donors Which dose of G-CSF is administered to the donors and at how many hours before donation? Do you combine G-CSF with steroids? If yes, at which dose and which kind of steroids – prednisolone or dexamethason (DXM)? If steroids are given alone, at how many hours before collection? prednisolone (dosage)? DXM (dosage)? Question 3 Frequency of donations Do you restrict the frequency of donations if the donor has been primed with G-CSF, steroids or both? What are your regulations concerning the accumulation of HES in the donor? Question 4 Sedimentation agents High- (Hetastarch, HS) and low molecular weight hydroxyethyl starch (Pentastarch PS) are used as sedimentation agents to enhance granulocyte collection. Which kind of hydroxyethyl starch is used at your institution for granulocyte collection? Question 5 Compatibility tests Do you respect or ignore the ABO and Rhesus match between donor and recipient? Do D-negative women younger than 50 years receive granulocytes only from D-negative donors? Question 6 Screening for leucocyte antibodies Because of the risk of TRALI, the ISBT working party has recommended to screen donor and recipient for the leucocyte antibodies. In case of positivity of either donor or recipient, a crossmatch between the plasma/serum of the positive individual and the leucocytes of the negative individual was recommended [4]. Do you screen or crossmatch the donor and recipient for leucocyte antibodies (HLA and HNA)? If positive, which consequences are drawn? Do you routinely reduce the plasma donations from female donors? In the USA, G-CSF is not approved by the FDA to be given to apheresis donors for mobilizing granulocytes. Furthermore, ‘granulocytes apheresis’ are not listed as an FDA-licensed product. In three of the four responding USA centres, the dose of G-CSF given to all donors before each collection is a standard one of 480 μg, whereas it is 600 μg in the fourth centre. In all centres, G-CSF is combined with oral dexamethasone (8 mg). In contrast, six European centres give varying doses of G-CSF according to the donors’ body weight, and only two apply a standardized dose of 300 μg G-CSF to all donors. Further, some European centres use only steroids for donor stimulation, while others use G-CSF alone. Although steroids alone are used by some of the other centres in the USA, they are not used alone by the four responding centres because granulocyte yields are substantially lower than those when donors are stimulated with combined G-CSF + dexamethasone. Moreover, the frequency of donations per donor is significantly less frequent in Europe than in the USA. Another difference is that in the USA, high molecular weight HES is used in three of the four centres because granulocyte yields are superior when compared to lower molecular weight HES, whereas this higher molecular weight HES is applied in only three European centres because of preference in Europe for the lower molecular weight formulation of HES. In two of the three USA centres that answered the question concerning ABO and Rh-D compatibility, ABO compatibility is required – similarly to Europe, where it is required in all but one centre. In two of the four USA centres, Rh-D compatibility is required. Screening donors for HLA and/or HNA antibodies is rarely performed, either in the USA or Europe. The apparent variation in methods for collecting granulocyte concentrates by apheresis requires interested readers to study all available reports and then to select the procedure that best suits their practice. This challenging field is certainly open for further research, and this IF may serve as a stimulating basis for further investigations. Guest Editor Ronald G. Strauss Professor Emeritus of Pathology & Pediatrics University Of Iowa College of Medicine Tel.: 1-319-338-8071 Fax: 1-319-356-0331 E-mail: ude.awoiu@ssuarts-dlanor International Forum Editors Simon Panzer University of Vienna Department Blood Group Serology and Transfusion Medicine Waahringer Gurtel 18-20 A-1090 Vienna, Austria E-mails: ta.ca.neiwinudem@reznap.nomis; ln.tenalpnpk@muroflanoitanretni and Henk W. Reesink Academic Medical Center Department Gastroenterology and Hepatology Amsterdam The Netherlands E-mails: ln.tenalpnpk@muroflanoitanretni; ln.cma@kniseer.w.h

BACKGROUND: Anemia is a serious problem in critically ill neonates. To investigate the pathophysiology of anemia and responses to red blood cell (RBC) transfusions and erythropoietin therapy, repeated measurement of red blood cell volume (RCV) and blood volume is useful. To extend our previous sheep study in which RBCs were labeled at four different biotin densities, we assessed the validity of this multidensity method for in vivo measurement of circulating RCV in humans. STUDY DESIGN AND METHODS: In eight healthy adults, autologous RBCs were biotinylated at each of four biotin densities (6, 18, 54, and 162 µg biotinylation reagent/mL RBC), mixed, and infused intravenously; blood was sampled at 10, 20, and 60 minutes. At each time, RCV was calculated from dilution of individual RBC populations enumerated by flow cytometry. RCV measurements from the population of RBCs biotinylated at 6 µg/mL were chosen as the reference values because this density had been previously validated against the 51Cr method in vitro and in vivo in humans. RESULTS: Values for RCVs were not significantly different among the four densities of biotinylated RBCs at any of the three time points and did not change over 60 minutes. CONCLUSIONS: These studies provide evidence that four densities of biotinylated RBCs can be used in vivo for simultaneous, independent, accurate measurements of RCV in humans. We speculate that this method will also be useful for repeated measurement of RCV and blood volume in infants and other patient populations in whom radioactive labels should be avoided.

BACKGROUND Safe, accurate methods permitting simultaneous and/or repeated measurement of red blood cell (RBC) survival (RCS) are important to investigate pathophysiology and therapy of anemia. Methods using chromium 51 (51Cr) -labeled RBCs are unacceptable for infants, children, and pregnant women. We report RCS measured in vivo using RBCs labeled with several densities of biotin (BioRBCs).

A “benchmark” is a standard or measure by which others can be assessed and/or compared. Benchmarking is the process of gathering information (ie, data and/or expert opinions) to set the benchmark. The greater the care, precision and validation taken during the benchmarking process, the more accurately the benchmark will reflect the optimal practices/issues in question and, in turn, allow the best point of reference for comparing/judging/instructing/advising others. In this issue of Transfusion, Fung et al report results of a benchmarking survey performed by the University HealthSystem Consortium (UHC) to assess the acceptability of various RBC anticoagulant/storage solutions and to determine the existence, or lack thereof, of policies to avoid possible hyperkalemia during RBC transfusions given to infants at member institutions.(1) The authors state in the title that this is a “benchmarking study” — which by convention implies that the results will serve as a standard by which others may be measured and/or as a point of reference from which other measurements can be made. Although the need to establish benchmarks for the issues raised (ie, RBC anticoagulant/storage solutions for infant transfusions and policies to avoid potential problems with transfusion-induced hyperkalemia) are well stated in the introduction by Fung et al,(1) the limitations/shortcomings of the benchmarking survey are multiple (as pointed-out by the authors in the discussion section). Thus, the extent to which the findings, conclusions and recommendations of Fung et al should be directly applied to neonatal/infant transfusion practices can be questioned. The basis for my opinion pertaining to limitations/shortcomings, is summarized by the following critical points — many of which were mentioned by the authors themselves,(1) but warrant emphasis here. First, response to the survey by institutions surveyed was only modest (47 of 107 UHC members = 44% for the RBC anticoagulant/preservative section and 45 of 107 = 42% for the hyperkalemia section), with no explanation offered as to why nonresponders failed to reply — other than mentioning that only 77 UHC members are “benchmarking program members” and noting that “no attempt was made at increasing response rate” beyond a few modest efforts.(1) Because UHC members are academic medical centers, one might assume that their practices would be as “evidence-based” as possible. Accordingly, to avoid possible misinformation due to reporting bias, more efforts should have been made to increase response rates to get a complete and accurate picture of member practices — to establish benchmarks that most likely reflect best/optimal practices. Second, one should question whether the most knowledgeable people completed the survey. The respondents were not neonatologists/pediatricians and only 21% were transfusion/pathology physicians. The remaining 79% of respondents were supervisors/managers/administrators.(1) Although these non-physician respondents can review blood bank/laboratory policies/procedures to answer survey questions, it is likely that they will not have in depth knowledge of transfusion practice guidelines/policies of the Departments of Pediatrics and Neonatology Divisions or of the Departments of Nursing. Moreover, they likely will not be familiar with the preferences/practices of physicians prescribing RBC transfusions for infants — particularly pertaining to potassium concerns. Third, to extend the second point, collecting data for which RBC anticoagulant/preservative solutions are “acceptable” provides only limited benchmarking data. For purposes of planning educational programs and recommending evidence-based guidelines/practices, it would be much more informative to know which anticoagulant/preservative solutions are “preferred” and, in the many institutions accepting multiple solutions, what percentage of RBC transfusions are actually given with the “accepted” anticoagulant/preservative solutions. In 19 of 47 responding institutions, four or five different anticoagulant/preservative solutions were “acceptable” with no indication as to which one(s) the prescribing physicians preferred and/or to what extent RBCs stored in different solutions were transfused. Simply acknowledging a solution is “acceptable” does not mean it is preferred or even actually used at an institution. Fourth, no mention is made as to how, or even whether, the survey answers were validated for completeness or accuracy. The authors stated that although no formal validation was performed, all participating UHC members were given opportunities to review and make corrections after close of the survey.(1) It would have increased credibility to report the number/percentage of UHC members who actually did review the completed survey and made corrections — and, in particular, to document whether answers to the survey were ever reviewed by physicians and nurses actually involved in RBC transfusions given to infants. Regarding the acceptability of anticoagulant/preservative solutions, the authors state that the decision to transfuse RBCs stored in additive solutions (AS-1, AS-3, AS-5) should be made by the medical director of the transfusion service, in consultation with institutional neonatologists — a point with which all would agree. The authors then make a rather feeble case for recommending RBCs stored in additive solutions as the best practice for low volume (

BACKGROUND: To investigate the pathophysiology of anemia and responses to red blood cell (RBC) transfusions and erythropoietin, repeated measurement of the circulating red blood cell volume (RCV) would be useful. Ovine erythropoiesis is similar to human erythropoiesis. Accordingly, a method for measuring RCV using either human or sheep RBCs labeled at different biotin densities has been previously validated in vitro. Here preclinical studies validating this method for in vivo measurement of circulating RCV in sheep are reported. STUDY DESIGN AND METHODS: For each sheep, autologous RBCs were biotinylated were at four discrete densities (12, 24, 48, and 96 µg biotinylation reagent/mL RBCs). The densities were mixed and infused intravenously. Blood was sampled five times over 1 hour beginning at 4 minutes. RCV values were determined based on dilution of each population of biotinylated RBCs and by the [14C]cyanate method. RESULTS: There was no difference among RCVs measured at all densities through 16 minutes; however, by 60 minutes, RBCs biotinylated at the highest density overestimated RCV by 7.6%. RCV values increased 41% over the hour, consistent with equilibration with a pool of RBCs sequestered in the spleen. RCV by the [14C]cyanate method paralleled results by the biotin method but averaged 8% greater. CONCLUSIONS: These studies provide evidence that all four densities of biotinylated RBCs can be used in sheep to simultaneously and independently determine RCV. We speculate that the multidensity biotinylation method will be useful both for multiple simultaneous measurements and for repeated measurement of circulating RCV and blood volume in humans.

Because most extremely preterm infants with birth weight less than 1000 g need red blood cell transfusions, many attempts have been made to collect, process, and store placental blood (ie, umbilical cord blood) for autologous transfusions. Although it is feasible to do this, multiple problems in doing so including insufficient volumes collected, clotting, hemolysis, bacterial contamination, failure to significantly supplant need for allogeneic transfusions, and high costs have led many to question whether, on balance, autologous/placental red blood cell transfusion offers clinically significant benefits.

BACKGROUND: Many clinical practices in transfusion medicine are controversial and/or lack definitive guidelines established by sound clinical trials. Although recommendations based on results of clinical trials performed using infants and children may not always be applied directly to adults—and vice versa—lessons learned from pediatric trials can be useful when critically assessing the design/results/conclusions of adult trials. STUDY DESIGN AND METHODS: Four randomized clinical trials (RCTs) studying pediatric patients were critically reviewed. They addressed two red blood cell (RBC) transfusion issues: 1) transfusion guidelines by which RBC transfusions are “triggered” by liberal (LIB; high pretransfusion patient hematocrit [Hct] levels) versus being “triggered” by restricted (RES; low pretransfusion Hct levels) and 2) transfusion of fresh RBCs (≤7 days' storage) versus RBCs (up to 42 days' storage). RESULTS: Findings established by primary outcomes generally were firm (e.g., fewer RBC transfusions were given to infants/children managed by RES guidelines; transfusing small volumes of RBCs stored up to 42 days to preterm infants diminished allogeneic donor exposures and were equally efficacious and safe as fresh RBCs stored ≤7 days). Findings based on secondary outcomes, subset, and post hoc analyses were inconsistent (e.g., clinical outcomes were equivalent after LIB or RES transfusions in only two of three RCTs; in the third, more neurologic problems were found in neonates given RES transfusions). CONCLUSIONS: Clinical practices should be based on data pertaining to the primary outcomes of RCTs, because trials are designed and statistically powered to address these issues. Clinical practices suggested by analysis of secondary outcomes, subsets of patients, and post hoc analyses should be applied cautiously until studied further—ideally, as primary outcomes in subsequent RCTs.

Many aspects of hematopoiesis are either incompletely developed in preterm infants or still functioning to serve the fetus (i.e., the intrauterine counterpart to a liveborn preterm neonate). This delayed development and/or slow adaptation to extrauterine life diminishes the capacity of the neonate to produce red blood cells (RBCs), platelets (PLTs), and neutrophils—particularly during the stress of life-threatening illnesses encountered after preterm birth such as sepsis, severe pulmonary dysfunction, necrotizing enterocolitis, and immune cytopenias. The serious medical and/or surgical problems of preterm birth can be further complicated by phlebotomy blood losses, bleeding, hemolysis, and consumptive coagulopathy. To illustrate, some preterm infants, especially those with birth weight less than 1.0 kg and respiratory distress, are given numerous RBC transfusions early in life owing to several interacting factors. Neonates delivered before 28 weeks of gestation (birth weight

Blood banks require a sensitive, specific, and efficient method to detect clinically significant RBC antibodies. Solid phase antibody screening methods are popular due to high sensitivity and automation. However, the high degree of reactivity detects "false positive" antibodies of questionable clinical significance leading to additional testing. We studied positive rates of Capture-R vs. PEG methods and categorized RBC antibodies identified by initial test results of 33,564 consecutive samples by Capture-R method. Capture-R was positive in 1,084/33,564 (3.2%) of samples. Using PEG as our "gold standard", PEG confirmed true positivity (i.e., > or = 1 cell reacting) in 710 Capture-R positive samples (65.5%); 374 Capture-R positive samples (34.5%) did not react in PEG (i.e., false positives). Of the 710 samples with true positivity, only 510 showed clinically significant alloantibodies. Using PEG as our "gold standard", only 2/3 of reactions by Capture-R were considered true positives. Because of ease and automation, Capture-R is popular as a screening test, but a more specific method may be helpful in order to identify truly significant alloantibodies.

Background A photochemical treatment (PCT) system has been developed to inactivate a broad spectrum of pathogens and white blood cells in platelet (PLT) products. The system comprises PLT additive solution (PAS III), amotosalen HCl, a compound adsorption device (CAD), a microprocessor-controlled ultraviolet A light source, and a commercially assembled system of interconnected plastic containers. Study design and methods A clinical prototype of the PCT system was used in a large, randomized, controlled, double-blind, Phase III clinical trial (SPRINT) that compared the efficacy and safety of PCT apheresis PLTs to untreated apheresis PLTs. The ability of multiple users was assessed in a blood center setting to perform the PCT and meet target process specifications. Results Each parameter was evaluated for 2237 to 2855 PCT PLT products. PCT requirements with respect to mean PLT dose, volume, and plasma content were met. Transfused PCT PLT products contained a mean of 3.6 x 10(11) +/- 0.7 x 10(11) PLTs. The clinical process, which included trial-specific samples, resulted in a mean PLT loss of 0.8 x 10(11) +/- 0.6 x 10(11) PLTs per product. CAD treatment effectively reduced the amotosalen concentration from a mean of 31.9 +/- 5.3 micromol per L after illumination to a mean of 0.41 +/- 0.56 micromol per L after CAD. In general, there was little variation between sites for any parameter. Conclusions The PCT process was successfully implemented by 12 blood centers in the United States to produce PCT PLTs used in a prospective, randomized trial where therapeutic efficacy of PCT PLTs was demonstrated. Process control was achieved under blood bank operating conditions.

Many controversial questions regarding the practice of neonatal red blood cell (RBC) transfusions exist, so that practices and policies vary widely. This article will critically assess information pertaining to two of these controversies, namely, the transfusion of RBCs stored for up to 42 days after collection vs the transfusion of fresh RBCs stored 7 days or less after donation and the use of recombinant human erythropoietin (rHuEPO) in attempts to either diminish the severity of or to treat the anemia of prematurity. Based on both theoretical considerations and several published clinical trials, RBCs from one donor stored up to 42 days in extended storage preservative solutions can safely provide all RBCs needed by most infants for small-volume transfusions. Based on a large number of clinical trials and a meta-analysis of these trials, it is impossible to provide firm guidelines for the use of rHuEPO in the treatment of the anemia of prematurity. Clearly, rHuEPO has efficacy in stimulating erythropoiesis in preterm infants, but success in the elimination or marked reduction in the need for RBC transfusions has not been definitively demonstrated.

BACKGROUND: The SPRINT trial examined efficacy and safety of photochemically treated (PCT) platelets (PLTs). PCT PLTs were equivalent to untreated (control) PLTs for prevention of bleeding. Transfused PLT dose and corrected count increments (CIs), however, were lower and transfusion intervals were shorter for PCT PLTs, resulting in more PCT than control transfusions. PLT dose was analyzed to determine the impact of the number of PLTs transfused on transfusion requirements. STUDY DESIGN AND METHODS: Transfusion response was compared for patients with all doses of ≥3.0 × 1011 and the complementary subset of patients with any dose of fewer than 3.0 × 1011. Analyses included comparison of bleeding, number of PLT and red blood cell (RBC) transfusions, transfusion intervals, and CIs between PCT and control groups within each PLT dose subset. RESULTS: Mean PLT dose per transfusion in the PCT group was lower than in the control group (3.7 × 1011 vs. 4.0 × 1011; p

BACKGROUND: Uncommonly, antibodies that appear to exhibit antigenic specificity on red blood cell (RBC) panels fail to maintain specificity following alloadsorption (i.e., they mimic antigenic specificity). Understanding both the pitfalls and the proper pathways to establish the diagnosis and to interpret the clinical significance of these mimicking antibodies is important for patient management. CASE REPORT: A 68-year-old woman was admitted with dyspnea, anemia, bilateral pulmonary emboli, and metastatic ovarian cancer. Blood bank evaluation identified anti-E reactivity in the patient's plasma sample and a positive direct antiglobulin test (DAT). RESULTS: The DAT was positive for immunoglobulin G and negative for C3b. An eluate of the RBCs showed E-antigen specificity on a RBC antibody panel. Repeat serologic testing with RBC antibody panels with adsorbed patient plasma showed removal of apparent anti-E reactivity with either E-antigen-positive or E-antigen-negative RBC stroma. CONCLUSION: A mimicking autoantibody with apparent E-antigen specificity was identified in the plasma sample of a woman with newly diagnosed ovarian cancer. Despite their relative low frequency, mimicking antibodies, whether auto- or alloantibodies, may interfere with the timely issuance of compatible blood products and may confuse laboratory and clinical staff. Determining the clinical significance of the antibody, by taking into account the RBC phenotype of the patient and the antigen prevalence in the general population, guides the extent of workup required to best utilize resources while assuring patient safety.

Summary Because plasma levels of erythropoietin are low in infants with the anemia of prematurity, it is logical to hypothesize that treatment with recombinant human erythropoietin (rHuEPO) would increase erythropoiesis and markedly diminish need for transfusions. Despite a large number of controlled clinical trials, the role of rHuEPO in neonatology practice is controversial. Clearly, it increases erythropoiesis in neonates and infants, but its efficacy in diminishing transfusions has been inconsistent, particularly in sick premature infants sustaining large phlebotomy loss for laboratory testing. Moreover, the ability to limit transfusions and donor exposures via modern transfusion practices coupled with concerns that the complete potential for adverse effects of rHuEPO is undefined have dissuaded widespread acceptance of rHuEPO as standard therapy to prevent or treat the anemia of prematurity.

Background The feasibility, efficacy, and safety of transfusing stored allogeneic RBCs has been demonstrated for small-volume transfusions given to infants. We measured the posttransfusion recovery and intravascular survival of allogeneic RBCs stored up to 42 days to further elucidate their efficacy.

BACKGROUND: The extend potential applications of a nonradioactive method for measuring circulating RBC volume, we tested the hypothesis that RBC volume could be determined independently using two populations of RBCs labeled with low-density biotin (LDB1) and high-density biotin (HDB). STUDY DESIGN AND METHODS: In 10 healthy adults, autologous RBCs were labeled with HDB, LDB, or 51Cr. The labeled RBCs were mixed and transfused. RBC volume was measured in postinfusion peripheral venous blood by quantitating dilution of each population of labeled RBCs. RESULTS: RBC volume measured using either LDB or HDB cells agreed well with RBC volume measured using 51Cr. For the regression of RBC volume by LDB versus RBC volume by 51Cr, correlation = 0.994 and slope = 0.933. For HDB versus 51Cr, correlation = 0.982 and slope = 0.953. RBC volume measured a second time in four subjects with HDB agreed well; mean CV for the differences between HDB and 51Cr were less than 5 percent. CONCLUSIONS: Using RBCs labeled with two different densities of biotin, RBC volume can be accurately measured simultaneously and repeatedly in the same subject without radiation exposure.

Little is known about the mechanisms explaining the wide variation in platelet counts (PLT) and other hematologic parameters in humans. We previously showed that the sex-based difference in hematocrit was associated with nucleotide variation in the erythropoietin receptor gene (EPOR). We sought to identify new polymorphisms of the human thrombopoietin (TPO) and thrombopoietin receptor (TPOR) genes to determine any associations with blood PLT counts. We screened TPO and TPOR for polymorphisms using single-strand conformation polymorphism (SSCP) and DNA sequencing. Association of polymorphisms was studied in 304 normal subjects with low or high PLT counts. Distribution of allelic frequency was analyzed by the Chi-square statistic. Single nucleotide polymorphisms (SNPs) with two alleles were found in TPO and TPOR. The TPO SNP was a G to A transition at nucleotide 5753, and the TPOR SNP was a C to A transversion at position 550 in the 5'-promoter area. The allelic frequencies were 0.54 for G and 0.46 for A of TPO, and 0.62 for C and 0.38 for A of TPOR in a Caucasian population. The frequency of the TPOR allele "C" was significantly higher in subjects with high PLT count (258 k/mm3) versus low PLT count (224 k/mm3) and in males with high PLT count (258 k/mm3) versus males with low PLT count (212 k/mm3). In contrast, the frequency of the TPO alleles was not related to blood PLT counts. An association of TPO and TPOR allele distribution to red and white blood cell parameters was seen. These new SNPs found for the human TPO and TPOR genes help explain variations in blood PLT counts and may be useful in patient studies related to the roles of TPO and/or TPOR in disease.

BACKGROUND: One problem assessing the hematologic physiology of preterm infants after delivery and/or the efficacy and toxicity of therapeutic interventions affecting RBC measurements is the inability of blood Hct values to accurately reflect circulating RBC volume—owing to changes in plasma volume that influence Hct (i.e., a fall in plasma volume concentrates RBCs to increase Hct; a rise in plasma volume dilutes RBCs to decrease Hct). STUDY DESIGN AND METHODS: As part of a randomized, clinical trial testing the hypothesis that delayed clamping of the umbilical cord at delivery expands neonatal circulating RBC volume, blood Hct was compared to circulating RBC volume results measured directly with autologous, biotinylated RBCs or estimated mathematically with neonatal body weight and Hct values in neonates after immediate or delayed (60 sec) cord clamping. RESULTS: Circulating RBC volume measured directly with biotinylated RBCs significantly increased (p=0.04) in neonates after delayed (42.1 ± 7.8 mL/kg) versus immediate (36.8 ± 6.3 mL/kg) cord clamping—a difference not detected indirectly by measuring Hct or estimating circulating RBC volume mathematically. CONCLUSIONS: Because true hematologic effects of delayed versus immediate cord clamping may not be apparent or may be misinterpreted, when based on indirect measurements of Hct or calculations of circulating RBC volume, it is important to measure circulating RBC volume directly—as done with autologous, biotinylated RBCs—to document whether delayed cord clamping truly results in a transfer of significant quantities of RBCs from placenta to neonate. The clinical benefits and potential toxicities of increased RBC transfer to neonates require further studies.

Modern blood separators rapidly process many liters of donor blood and efficiently collect vast quantities of blood components from donors, who may be stimulated with potent recombinant hematopoietic growth factors or cytokines. Accordingly, the potential risks of modern multiple product/unit apheresis donations and recombinant growth factors is analyzed in this report. As is true for all medical procedures, risks are associated with apheresis donations. Risks of a "standard" apheresis donation, in which one unit of PLTs or plasma is collected, are comparable to the risks of whole blood donation. Risks of multiple unit apheresis donations, in which either vast quantities of a single blood component or multiple units of various components are collected, are incompletely understood, particularly, when donors are stimulated with recombinant hematopoietic growth factors to increase component yields. To minimize donor risks and to increase knowledge of multiple component apheresis donations, both short-term problems (e.g., donor reactions accompanying apheresis procedures and pre- vs. post-procedure changes in results of donor laboratory studies) and long-term problems (e.g., medical diagnoses/problems and abnormalities of donor blood counts and laboratory test results) should be monitored, ideally, by a repeat donor registry. When recombinant hematopoietic growth factors are prescribed, donors should give informed consent, and blood center professionals must be aware of 1) the effects of these drugs given at pharmacologic, rather than physiologic, doses; 2) the differences between the molecular structure of recombinant vs. natural/endogenous growth factors; 3) the fact that recombinant growth factors have both narrow/focused and broad biological activities; and 4) the probability that results of studies in sick/immunosuppressed patients may not be applicable to healthy/immunocompetent donors.

We are writing regarding the following comment by Oliver and colleagues1 in the August issue of TRANSFUSION. The authors state in the Introduction that “Radioisotope chromium 51 (51Cr) labeling of blood, despite its disadvantages, is still the only reliable method to predict the intravascular success of an incompatible transfusion. Alternative techniques such as direct biotinylation of red blood cells (RBCs), despite their promise, have not been routinely adopted.” We concur that biotinylated red blood cells (RBCs) have not yet been routinely adopted for quantitation of RBC survival, due in part to the current Food and Drug Administration requirement that an investigational new drug is required for studies that include transfusion with biotin-labeled RBCs. Nonetheless, several factors lead us to predict that measurement of RBC survival using the biotin label will be considered to serve as the reference method eventually. More recent publications than those cited by Oliver and colleagues indicate that the biotin method for labeling RBCs is now reaching its promise. Human RBCs may be reliably biotinylated ex vivo. Ten to 50 mL of labeled RBCs administered to a 70-kg adult yield 0.4% to 2% biotinylated RBCs in the bloodstream; this initial percentage is adequate to permit accurate tracking by flow cytometry for greater than 85% of RBC survival.2,3 In addition to basic measures of RBC recovery and life span, the biotin method has been used in humans for studies to compare survival of multiple RBC populations concurrently in the same individual and to assess age-dependent changes in RBCs, including the following: The survival characteristics of two populations of RBCs that were labeled with different biotin densities and were distinguishable by flow cytometry were simultaneously studied in the same study subject.2 The 51Cr RBC label can be used for only a single population of RBCs at any one time and, consequently, studies of multiple RBC populations must be done sequentially—possibly, introducing biologic and/or temporal variability because studies were not done concurrently. Time-dependent changes in the properties of sickle RBCs have been determined, including the survival of biotinylated RBCs containing HbF versus those without detectable HbF.4 The rate of formation of glycated Hb (e.g., HbA1c) in controls and patients with diabetes determined using magnetic isolation of aging biotin-labeled RBCs has been helpful in interpreting the biologic and clinical significance of glycated Hb as an indicator of high blood glucose levels and long-term complications of diabetes.5 In studies such as those by Mock and colleagues2 and Franco,3 understanding intrasubject variability in RBC survival is important. The accuracy of the 51Cr method is limited due to dissociation from the beta-chain of Hb in the RBCs at rates that vary substantially among individuals and even more among species. This loss of label has a direct effect on accuracy of RBC life span quantitation determined with 51Cr. The situation is different with biotinylated RBCs, even though up to 40% of the covalently linked biotin labels on the outward facing membrane of the RBC are lost during the first few weeks in the circulation. Because the biotinylated RBCs are examined and enumerated individually by flow cytometry and continue to fall within the designated region for a positive cell, they are counted accurately. Accordingly, RBC recovery and life span can be quantitated until nearly all of the biotin-labeled RBCs are removed from the circulation, that is, until the biotin-labeled RBCs fall below 0.06% of the circulating RBCs. The biotin RBC label has been used successfully in all mammals tested thus far and is thus useful in preclinical as well as clinical studies. Current information indicates that biotinylated RBCs may be used safely. In adult humans, the biotin RBC label has been associated with the development of transient agglutinating antibodies to biotin-labeled RBCs in approximately 15% of adult study subjects infused with 50 mL of biotinylated RBCs.6 In the 12 years since the publication of these observations, we have observed only one of eight (13%) additional adult participants who developed a transient positive direct antibody result (Table 1).2 Of note, in this more recent report, subjects were infused with four times the volume of biotinylated RBCs (200 mL) containing an estimated sevenfold greater biotin density per RBC without an increase in the percentage of individuals developing antibodies. The development of antibodies to biotin-labeled RBCs had no effect on the survival of biotin-labeled RBCs. The four adult subjects who developed antibodies to biotinylated RBCs have not developed an identifiable change in the rate of elimination of biotin-labeled RBCs,2,6 clinical signs, or symptoms (personal follow-up communication with study subjects). However, we note that none of these four individuals have been rechallenged with biotinylated RBCs. TABLE 1 Time course after transfusion of antibodies to biotinylated RBCs We write to call these more recent studies utilizing biotin labeling of RBCs to the attention of the authors and other investigators performing RBC kinetic studies so that the advantages of biotin-labeled RBCs can be exploited.

HES solutions provide a sterile, alternative colloidal fluid to albumin solutions and/or plasma in the management of patients who need plasma volume expansion. Solutions of HES are widely accepted internationally but are used only modestly in the United States, largely because of concerns over hemostasis.A randomized, blinded, two-arm trial comparing the hemostatic effects of pentastarch versus hetastarch when infused in the clinically relevant dose of 90 g of HES dissolved in 1.5 L of saline was conducted. Multiple studies of fibrin clot formation, fibrinogen/fibrinolysis, and platelet (PLT) functions were performed before and on multiple occasions for 70 days following HES infusion.Several significant abnormalities of hemostasis assay results occurred following HES infusions, with hetastarch causing significantly greater abnormalities than pentastarch. Individual clotting proteins and blood PLTs fell modestly because of plasma volume expansion and hemodilution. A fall in excess of that caused by hemodilution was demonstrated for von Willebrand factor antigen plus its associated FVIII and ristocetin cofactor activities. The partial thromboplastin time was prolonged, whereas the thrombin time was shortened. Plt function abnormalities were seen in most subjects to a modest degree. Studies of fibrinolysis were normal.Solutions of hetastarch produce significant abnormalities of some hemostasis laboratory results when infused at clinically relevant doses, but it is unlikely that the modest hemostatic abnormalities produced at these doses per se would lead to clinical bleeding. Hetastarch causes greater hemostatic abnormalities than pentastarch, and because both HES solutions have comparable plasma volume-expanding effects, it is reasonable to prefer pentastarch as a plasma volume expander.

A survey was completed by 25 medical directors at different institutions performing plateletpheresis. The practices of these 25 physicians were analyzed regarding the acceptance/rejection of plateletpheresis donors with a history of cardiac disease/surgery, seizures/epilepsy, cancer, or autoimmune diseases. Although available medical literature documents little risk of these disorders either to donors (i.e., donation reactions) or to transfusion recipients (i.e., disease transmission), up to 24% of medical directors outright reject some of these potential donors while others accept patients/donors with these illnesses, providing they meet certain medical/health criteria. Acceptance/rejection of individuals with medical disorders has relevance for the availability of the blood supply and blood product shortages because several million Americans, diagnosed with these illnesses, represent a sizable pool of potential blood and platelet donors.

At one blood center, each of 20 donors underwent plateletpheresis on four blood cell separators in random order. We compared the CS3000+, Amicus V 2.41, MCS Plus, and Spectra LRS V 7 Turbo regarding platelet (PLT) yield, pre- and post-procedure PLT counts, percent fall in donor PLT count, process time, efficiency, PLT product and donor PLT volume (MPV). Using ≥ 150 × 109 PLTs/L pre-donation counts, a goal was set of 4.5 × 1011 PLTs unit in up to 100 minutes processing time. Results were (mean values) PLT yields of Amicus, Spectra, CS3000+, and MCS Plus: 4.3, 4.6, 4.3, 4.0 × 1011 PLTS, respectively; percent donor PLT fall: 24, 32, 30, 29%, respectively; processing times: 50, 74, 87, 101 minutes, respectively; relative efficiency (RE): 2.2, 1.6, 1.2,1.0, respectively (based on the MCS Plus performance with RE of 1 = 4 × 109 PLTS/min); PLT product MPV: 6.7, 7.4, 6.8,7.1 fL, respectively; pre-procedure donor MPV: 7.7, 7.3, 7.6 and 7.6 fL, respectively; and percent donor MPV change: −5.2, 0, −6.6, and −10%, respectively. Significant changes in the donor MPV were noted (P < 0.05) but could not be related to product MPV. Spectra seemed to collect larger PLTs (higher MPV); the significance remains unknown for both donors and recipients. Importantly, all four separators gave acceptable and comparable PLT yields (P < 0.05) with Spectra trending higher. The short process time and high RE together indicate highly efficient collections particularly by Amicus and Spectra. J. Clin. Apheresis 17:170–176, 2002. © 2002 Wiley-Liss, Inc.