Blood counts in children with Down syndrome.

BACKGROUND: Children with Down syndrome (DS) are more likely to have hematologic and immunologic abnormalities compared to their typically developing peers, but normal ranges have not been defined. The goal of this study was to create references for complete blood counts (CBCs) in patients with DS. METHODS: A retrospective investigation of 355 (male = 196, 55.2%; mean age = 6.49 years, SD = 5.07) healthy pediatric patients with DS who received a CBC between 2011 and 2017 as part of their medical care at a single, large, pediatric teaching hospital. Control data on 770 healthy patients without DS were included. Descriptive statistics were performed on demographic and clinical characteristics. Kruskal-Wallis H tests, nested analysis-of-variance tests, and t-tests were run to determine the significant associations. RESULTS: Age-related normative curves for healthy children with DS outlining 2.5th, 25th, 50th, 75th, and 97.5th percentiles are provided for total white blood count, hemoglobin concentration, hematocrit, mean corpuscular volume, and platelet, absolute neutrophil, absolute lymphocyte, eosinophil, monocyte, and basophil counts. Statistical differences were found between children with and without DS receiving care at the same hospital based on matched age/sex groups. CONCLUSIONS: This study demonstrates that patients with DS have different reference ranges for multiple blood counts compared to those without DS, creating a new resource for pediatricians to refer to when evaluating CBCs in this population.

Effect of cord blood processing on transplantation outcomes after single myeloablative umbilical cord blood transplantation.

Variations in cord blood manufacturing and administration are common, and the optimal practice is not known. We compared processing and banking practices at 16 public cord blood banks (CBB) in the United States and assessed transplantation outcomes on 530 single umbilical cord blood (UCB) myeloablative transplantations for hematologic malignancies facilitated by these banks. UCB banking practices were separated into 3 mutually exclusive groups based on whether processing was automated or manual, units were plasma and red blood cell reduced, or buffy coat production method or plasma reduced. Compared with the automated processing system for units, the day 28 neutrophil recovery was significantly lower after transplantation of units that were manually processed and plasma reduced (red cell replete) (odds ratio, .19; P = .001) or plasma and red cell reduced (odds ratio, .54; P = .05). Day 100 survival did not differ by CBB. However, day 100 survival was better with units that were thawed with the dextran-albumin wash method compared with the "no wash" or "dilution only" techniques (odds ratio, 1.82; P = .04). In conclusion, CBB processing has no significant effect on early (day 100) survival despite differences in kinetics of neutrophil recovery.

Hydroxyethyl starch and granulocyte transfusions: considerations of utility and toxicity profile for patients and donors.

Hydroxyethyl starch (hetastarch) is a synthetic glucose compound with extensive clinical use as a volume expander. Because of its red blood cell-sedimenting properties, hetastarch plays a major role in preparation of granulocyte products. Recent concerns have been raised about the use of hetastarch in critically ill patients for the development of renal injury and other severe adverse events. In contrast, granulocyte donors receive much less of this compound during collection procedures, and over many years, minimal toxicity has been documented in these individuals. Furthermore, granulocyte products contain very little hetastarch, and ill effects on renal function have not been associated with their administration. This review assesses available information about the toxicity profile of hetastarch in critically ill patients requiring a volume expander as well as granulocyte donors and recipients. Because of the lack of toxicity in these latter two groups, hetastarch should be available for preparation of granulocyte products and their administration.

Spectra Optia granulocyte apheresis collections result in higher collection efficiency of viable, functional neutrophils in a randomized, crossover, multicenter trial.

BACKGROUND: Granulocyte transfusion from healthy donors is used in the treatment of patients with granulocyte function defects, or transient neutropenia and severe bacterial or fungal infections resistant to maximal antimicrobial treatment. STUDY DESIGN AND METHODS: This study evaluated the performance and safety of the newly developed granulocyte collection protocol of the Spectra Optia in a prospective, multicenter, open-label, randomized, paired crossover trial compared with the COBE Spectra apheresis system in a population of 32 evaluable healthy subjects. All subjects received granulocyte-colony-stimulating factor and dexamethasone before collection. RESULTS: Granulocyte procedures from Spectra Optia apheresis procedures had an approximately 23% higher polymorphonuclear (PMN) collection efficiency (CE) than the COBE Spectra collections (mean, 53.7% vs. 43.2%; p < 0.01), while the platelet CE (10.9% vs. 10.8%, respectively) and hematocrit (7.4% vs. 7.4%) were comparable between collections on both devices. Spectra Optia collections generated a higher total PMN yield per liter of blood processed than those produced by the COBE Spectra (with means of 8.6 × 10(10) vs. 6.8 × 10(10) , respectively). Granulocyte viability was more than 99% with both devices, and chemotaxic and bacterial killing activities of circulating versus collected granulocytes were similarly preserved. Fewer operator adjustments were required with Spectra Optia and there was no significant difference in the number or intensity of adverse events between instruments. CONCLUSION: CE of the granulocyte collection procedure with the Spectra Optia was approximately 10 percentage points higher than with the COBE Spectra, required less operator involvement, and is safe for clinical implementation.

Red cell exchange transfusion halts progressive proliferative sickle cell retinopathy in a teenaged patient with hemoglobin SC disease.

A male with sickle SC disease presented at age 8 years with proliferative sickle cell retinopathy (PSCR) and bilateral vitreous hemorrhage which spontaneously resolved, then recurred at 13 years of age. Despite conventional therapy with repeated pan-retinal photocoagulation and pars plana vitrectomy, he developed progressive PSCR and recurrent vitreous hemorrhage over the next 30 months. We describe the successful use of chronic red cell exchange transfusion (RCE) to preserve his vision and stabilize the retinopathy.

One size will never fit all: the future of research in pediatric transfusion medicine.

There is concern at the National Heart, Lung, and Blood Institute (NHLBI) and among transfusion medicine specialists regarding the small number of investigators and studies in the field of pediatric transfusion medicine (PTM). Accordingly, the objective of this article is to provide a snapshot of the clinical and translational PTM research considered to be of high priority by pediatricians, neonatologists, and transfusion medicine specialists. Included is a targeted review of three research areas of importance: (i) transfusion strategies, (ii) short- and long-term clinical consequences, and (iii) transfusion-transmitted infectious diseases. The recommendations by PTM and transfusion medicine specialists represent opportunities and innovative strategies to execute translational research, observational studies, and clinical trials of high relevance to PTM. With the explosion of new biomedical knowledge and increasingly sophisticated methodologies over the past decade, this is an exciting time to consider transfusion medicine as a paradigm for addressing questions related to fields such as cell biology, immunology, neurodevelopment, outcomes research, and many others. Increased awareness of PTM as an important, fertile field and the promotion of accompanying opportunities will help establish PTM as a viable career option and advance basic and clinical investigation to improve the health and wellbeing of children.

Peroxiredoxin 6 translocates to the plasma membrane during neutrophil activation and is required for optimal NADPH oxidase activity.

Neutrophils provide the first line of defense against microbial invasion in part through production of reactive oxygen species (ROS) which is mediated through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generating superoxide anion (O2-). The phagocyte oxidase (phox) has multiple protein components that assemble on the plasma membrane in stimulated neutrophils. We recently described a protein in neutrophils, peroxiredoxin 6 (Prdx6), which has both peroxidase and phospholipase A2 (PLA2) activities and enhances oxidase activity in an SDS-activated, cell-free system. The function of Prdx6 in phox activity is further investigated. In reconstituted phox-competent K562 cells, siRNA-mediated suppression of Prdx6 resulted in decreased NADPH oxidase activity in response to formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). In neutrophils stimulated with PMA, Prdx6 translocated to plasma membrane as demonstrated by Western blot and confocal microscopy. Translocation of Prdx6 in phox competent K562 cells required both p67phox and p47phox. In addition, plasma membrane from PMA-stimulated, oxidase competent K562 cells with siRNA-mediated Prdx6 suppression contained less p47phox and p67phox compared to cells in which Prdx6 was not decreased. Cell-free oxidase assays showed that recombinant Prdx6 did not alter the Km for NADPH, but increased the Vmax for O2- production in a saturable, Prdx6 concentration-dependent manner. Recombinant proteins with mutations in Prdx (C47S) and phospholipase (S32A) activity both enhanced cell-free phox activity to the same extent as wild type protein. Prdx6 supports retention of the active oxidase complex in stimulated plasma membrane, and results with mutant proteins imply that Prdx6 serves an additional biochemical or structural role in supporting optimal NADPH oxidase activity.

Phox activity of differentiated PLB-985 cells is enhanced, in an agonist specific manner, by the PLA2 activity of Prdx6-PLA2.

Peroxiredoxin 6-phospholipase A(2) (Prdx6-PLA(2) ) is a bi-functional enzyme with peroxi-redoxin (Prdx) and phospholipase A(2) (PLA(2) ) activities. To investigate its impact on phagocyte NADPH oxidase (phox) activity in a neutrophil model, the protein was knocked down in PLB-985 cells using stable expression of a small hairpin RNA (shRNA) and phox activity was monitored after cell differentiation. The knockdown cells had reduced oxidase activity in response to stimulation with the formylated peptide fMLF, but the response to the phorbol ester PMA was unchanged. Reintroduction of shRNA-resistant Prdx6-PLA(2) into the knockdown cells by stable transfection with a Prdx6-PLA(2) expression plasmid restored the fMLF response, as did reintroduction of Prdx6-PLA(2) mutated in the Prdx active site; reintroduction of PLA(2) active site mutants, however, failed to restore the response. Thus, the PLA(2) activity of Prdx6-PLA(2) in intact cells mediates its ability to enhance phox activity in response to fMLF. In combination with previous publications by other groups, our work indicates that various PLA(2) isoforms can enhance oxidase activity but they are differentially important in different cell types and in the response to different agonists.

Sweet syndrome in an infant with chronic granulomatous disease.

Sweet syndrome is characterized by painful, erythematous cutaneous lesions containing neutrophilic infiltrates. Although more commonly seen in adults, Sweet syndrome has also been recognized in several pediatric cases. Two previous cases of pediatric Sweet syndrome and 1 adult case have been described in chronic granulomatous disease (CGD) patients. We report the case of an infant with known CGD who was presented with methicillin-sensitive Staphylococcus aureus lymphadenitis and subsequently developed Sweet syndrome. CGD patients are prone to several disorders of inflammation. This case illustrates that Sweet syndrome may be part of the spectrum of inflammatory conditions to which CGD patients are predisposed.

In vivo interferon-gamma induced changes in gene expression dramatically alter neutrophil phenotype.

The cytokine Interferon-γ (IFN-γ) exerts powerful immunoregulatory effects on the adaptive immune system and also enhances functions of the neutrophil (PMN). The clinical use of IFN-γ has been driven by the finding that its administration to patients with chronic granulomatous disease (CGD) results in decreased incidence and severity of infections. However, IFN-γ has no effect on the characteristic defect of CGD, the inability to convert oxygen to microbicidal metabolites including superoxide anion (O2-) during the phagocytosis associated oxidative burst. We administered varying doses of IFN-γ to adult volunteers and studied the effects on plasma drug levels and response molecules and PMNs isolated from blood drawn at intervals over a 96- hour period. Plasma concentrations of IFN-γ, IP-10 and neopterin, and stimulated release of O2- from PMNs exhibited dose- and time-dependent increases after IFN-γ administration. Gene expression in PMNs was altered for 2775 genes; changes occurred rapidly after administration and returned to baseline in 24-36 hours. Several genes involved with neutrophil host defense were upregulated including those for components of the O2- generating NADPH oxidase; innate-immune and Fc receptors; proteins involved in MHCI and II; a regulator of circulating PMN number; guanylate binding proteins; and a key enzyme in synthesis of an essential NOS cofactor. Coordinate changes were detected in protein levels of representative products from several of these genes. Lysates from isolated neutrophils also demonstrated a spike in NO following IFN-γ administration. IFN-γ appears to increase non-oxygen dependent microbicidal functions of PMNs which could provide strategies to compensate for deficiencies, explain its clinical benefit for CGD patients and expand therapeutic applications of IFN-γ to other disorders. Trial registration: Protocol registered in ClinicalTrials.gov, NCT02609932, Effect of IFN-γ on Innate Immune Cells.

Extended red blood cell antigen matching for transfusions in sickle cell disease: a review of a 14-year experience from a single center (CME).

BACKGROUND: Alloimmunization to red blood cell (RBC) blood group antigens is a major complication for patients with sickle cell disease (SCD), which limits the usefulness of RBC transfusion. Here, we report our experiences with extended RBC antigen matching for SCD patients. STUDY DESIGN AND METHODS: Records for 99 SCD patients transfused only with the extended matching protocol between 1993 and 2006 were reviewed. Patients and donors were phenotyped for 20 blood group antigens and RBC units that were negative for antigens not expressed by the recipient were provided. When necessary, mismatches were allowed at Le(a) , Le(b) , Fy(b) , and MNSs to meet requirements for antigens regarded as the most clinically significant. Matched RBC units (6946) were provided to 99 patients (mean, 70 units/patient; range, 1-519 units/patient). Eliminating mismatches, 90% of the transfusions matched all other negative antigens. RESULTS: Seven alloantibodies were detected in seven patients resulting in 7% alloimmunized at a rate of 0.1 antibodies per 100 units transfused. Three recipients who developed antibodies were D mosaic and would have been mistyped with serologic techniques. Alloimmunization was decreased compared to ABO and/or D matching at our institution and others. Twelve autoantibodies and no severe hemolytic transfusion reactions were reported. CONCLUSION: Exact matching for ABO, Rhesus, Kell, Kidd, and Fy(a) and extending this match whenever possible is an effective strategy to reduce alloimmunization to RBC antigens. Consideration should be given to exploring this conclusion further with a controlled, multi-institutional trial to determine efficacy, cost-benefit analysis, and reproducibility of this approach.

Generation of neutrophil priming activity by cell-containing blood components treated with pathogen reduction technology and stored in platelet additive solutions.

BACKGROUND: Storage of cell-containing blood components such as platelet concentrates (PCs) and red blood cells (RBCs) results in generation of biologically active compounds, many of which may be associated with adverse transfusion events. Priming of the neutrophil oxidase activity is a common characteristic of many of the biologically active compounds found in stored blood. We evaluated the priming activity of pathogen reduction technology (PRT)-treated PCs stored in plasma or platelet additive solution (PAS) and PRT-treated RBCs. STUDY DESIGN AND METHODS: PCs were collected with Trima or Amicus equipment and were PRT treated with the Mirasol PRT system or the Intercept Blood System. Some units were gamma irradiated. Products were stored in 100% plasma or 35% plasma plus PAS. RBCs were washed and PRT treated before storage. Samples were removed throughout storage and priming of the oxidase activity was measured. RESULTS: Platelets collected on Trima or Amicus equipment and stored in plasma or PAS demonstrated increasing priming activity during 5 to 7 days of storage. Gamma irradiation, but not PRT treatment with either technology, further enhanced this priming activity. Supernatants of RBCs stored for 42 days induced priming in untreated controls, but not in washed or Mirasol PRT-treated test products. CONCLUSION: Production of oxidase priming activity increased during storage in all blood products. No significant differences were associated with the collection method, storage in PAS, or PRT treatment. The generation of biologically active compounds, which may serve as an etiology for adverse events, appears to be independent of these processes for collection, storage, and pathogen reduction.

Consensus recommendations of pediatric transfusion medicine objectives for clinical pathology residency training programs.

BACKGROUND: Pediatric transfusion medicine (PTM) is a subspecialty of transfusion medicine with no formal training program and few specialists. The Pediatric Transfusion Medicine Academic Awardees (PedsTMAA) group surveyed PTM content experts to identify relevant objectives for the first formal PTM curriculum. STUDY DESIGN AND METHODS: Eight North American PTM experts were invited to participate in a two-step consensus process. PTM-related objectives compiled from a review of existing training documents were organized into a survey. Experts were asked to rate each objective for relevancy for a clinical pathology trainee. Content validity indexes (CVIs) and asymmetric confidence intervals (ACIs) of expert ratings and analysis of respondents' comments were used to identify relevant objectives. RESULTS: Six experts participated and reviewed 117 objectives. Based on content validity criteria (CVI > or = 0.83 and lower-limit 95% ACI > or = 3), a total of 65 objectives were considered relevant. Twenty-three objectives were rated "very relevant" by all the experts while some proposed objectives were determined to be not relevant, out of date, or inappropriate for a resident trainee level. CONCLUSIONS: The PedsTMAA group identified 65 objectives for a PTM curriculum. Twenty-three represent a clear core set of objectives and should be considered for clinical pathology training. The next step is to consider the teaching strategies and evaluation methods that will be employed to best deliver this content addressing competency in medical knowledge.

Long term G-CSF-induced remission of ulcerative colitis-like inflammatory bowel disease in a patient with glycogen storage disease Ib and evaluation of associated neutrophil function.

We present a 23-year-old female with Glycogen storage disease Ib (GSD Ib) who was diagnosed with ulcerative colitis-like inflammatory bowel disease (IBD) at 7 years of age. G-CSF therapy reversed the IBD, was required to maintain IBD remission and was well tolerated. Neutrophil functions at time of diagnosis showed impaired chemotaxis but normal superoxide anion production and bactericidal activity. Ulcerative colitis-like IBD may also be seen in GSD Ib and is responsive to G-CSF therapy. Neutrophil dysfunction is variable among patients with GSD Ib.

Metabolic abnormalities in G6PC3-deficient human neutrophils result in severe functional defects.

Severe congenital neutropenia type 4 (SCN-4) is an autosomal recessive condition in which mutations in the G6PC3 gene encoding for the catalytic 3 subunit of glucose-6-phosphatase-ß result in neutropenia, neutrophil dysfunction, and other syndromic features. We report a child with SCN-4 caused by compound heterozygous mutations in G6PC3, a previously identified missense mutation in exon 6 (c.758G>A[p.R235H]), and a novel missense mutation in exon 2 (c.325G>A[p.G109S]). The patient had recurrent bacterial infections, inflammatory bowel disease, neutropenia, and intermittent thrombocytopenia. Administration of granulocyte colony-stimulating factor (G-CSF) resolved the neutropenia and allowed for detailed evaluation of human neutrophil function. Random and directed migration by the patient's neutrophils was severely diminished. Associated with this were defects in CD11b expression and F-actin assembly. Bactericidal activity at bacteria/neutrophil ratios >1:1 was also diminished and was associated with attenuated ingestion. Superoxide anion generation was <25% of control values, but phox proteins appeared quantitatively normal. Extensive metabolomics analysis at steady state and upon incubation with stable isotope-labeled tracers (U-13C-glucose, 13C,15N-glutamine, and U-13C-fructose) demonstrated dramatic impairments in early glycolysis (hexose phosphate levels), hexosemonophosphate shunt (required for the generation of the NADPH), and the total adenylate pool, which could explain the dramatic cell dysfunction displayed by the patient's neutrophils. Preliminary experiments with fructose supplementation to bypass the enzyme block demonstrated that the metabolic profile could be reversed, but was not sustained long enough for functional improvement. In human deficiency of G6PC3, metabolic defects resulting from the enzyme deficiency account for diverse neutrophil functional defects and present a major risk of infection.

Lack of antibody formation to platelet neoantigens after transfusion of riboflavin and ultraviolet light-treated platelet concentrates.

BACKGROUND: Pathogen inactivation technologies provide a potential solution to donor screening and blood testing strategies reducing the risk of transfusion-transmitted infectious diseases. The Mirasol pathogen reduction technology (PRT) system (CaridianBCT) uses riboflavin and UV light to introduce modifications in nucleic acids, reducing the infectious pathogen load in blood components. This study evaluated serum of patients who received PRT-treated platelet (PLT) concentrates over a time period of 28 days for the appearance of antibodies to neoantigens on PLTs. STUDY DESIGN AND METHODS: Serum specimens were obtained at study inclusion and at the 28-day follow-up visit from patients randomly assigned to receive PRT-treated PLT concentrates and at study inclusion of control subjects receiving untreated PLTs. PLT samples from untreated and PRT-treated PLTs were collected. PLT samples for each patient were pooled for the analysis. The presence of antibodies in patient serum to neoantigens was determined with a modified Capture-P assay. The presence of auto- or alloantibodies in specific patient samples was determined with PAKAUTO and PAK 12 techniques. RESULTS: Forty-four patients receiving PRT-treated PLTs were evaluated; none of these patients demonstrated antibodies to neoantigens. One patient demonstrated a PLT alloantibody to human PLT antigen (HPA)-5b and autoreactivity to glycoprotein Ia/IIa, consistent with an alloantibody at the beginning, but not at the end of the study interval. Of 22 patients evaluated in the control group, one alloantibody with HPA-5b reactivity was detected. CONCLUSION: Patients receiving PRT-treated PLT concentrates did not demonstrate antibodies to neoantigens, suggesting that neoantigen formation is not a critical side effect of this pathogen reduction process.

Pediatric transfusion medicine: development of a critical mass.

Many significant events have occurred in the recent past that beg a broad audience to address the question "What is pediatric transfusion medicine?" Herein, we list some of these events and their relevance below and attempt to provide an answer for this question. Indeed, several issues regarding the subspecialty of pediatric transfusion medicine (PTM) are particularly timely, and it appears that a critical mass, or a nidus capable of becoming a critical mass, is developing in PTM.

The effect of storage on the accumulation of oxidative biomarkers in donated packed red blood cells.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a life-threatening condition characterized by oxidative stress. Longer storage times of packed red blood cells (PRBC) and other blood products have been implicated with an increased risk in developing TRALI in transfused patients. METHODS: A total of 10 units of blood containing PRBC stored in citrate-phosphate-dextrose buffer at 4 degrees C were included in the study. At Bonfils Blood Center (Denver, CO), samples were collected on storage day 1 and day 42. Samples were immediately centrifuged, and the supernatants were collected and stored at -80 degrees C until further analysis. Oxidation-reduction potential and protein oxidation were measured in both the day 1 and day 42 samples. RESULTS: Oxidation-reduction potential significantly increased (p < 0.05) in the day 42 sample (98.1 mV +/- 21.9 SD) versus the day 1 sample (62.6 mV +/- 21.5 SD). The oxidation of human serum albumin increased by 63.6% during the storage time. Other serum proteins such as apolipoprotein A1 and transthyretin demonstrated similar increases in oxidation. Also, proteins with a cleaved C-terminal amino acid were observed indicating the presence of carboxypeptidase activity, a marker of inflammation. CONCLUSIONS: The presence of an oxidative environment in transfused PRBC increases with storage time. This could partially explain the increased risk of developing TRALI related to the transfusion of older blood products.