Papers by Steven Spitalnik
Haematologica, Jan 27, 2017
Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, mos... more Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored RBC units is clinically relevant for transfused recipients. Leukoreduced RBCs from G6PD-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with SO2 ranging from <3 to >95%). RBCs from healthy human volunteers were also collected at sea level or after 1-7 days at high altitude (>5000m). Finally, C57BL/6J mouse RBCs were incubated in vitro with 13C1-aspartate or 13C5-adenosine under normoxic or hypoxic conditions, with or without deoxycoformicin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse RBCs stored for up to 42 or 14 days, respectively, and correlated with 24h post-transfusion RBC recovery studies. Hypoxanthine increased in stored RBC units as a fun...
Blood transfusion = Trasfusione del sangue, 2017
Most frequent red cell (RBC) donors and many first-time donors are iron deficient, but meet haemo... more Most frequent red cell (RBC) donors and many first-time donors are iron deficient, but meet haemoglobin standards. However, the effects of donation-induced iron deficiency on RBC storage quality are unknown. Thus, we used a mouse model to determine if donor iron deficiency reduced post-transfusion RBC recovery. Weanling mice received a control diet or an iron-deficient diet. A third group receiving the iron-deficient diet was also phlebotomised weekly. This provided 3 groups of mice with different iron status: (1) iron replete, (2) mild iron deficiency with iron-deficient erythropoiesis, and (3) iron-deficiency anaemia. At ten weeks of age, blood was collected, leucoreduced, and stored at 4 ºC. After 12 days of storage, 24-hour (h) post-transfusion RBC recovery was quantified in recipients by flow cytometry. Before blood collection, mean haemoglobin concentrations in the iron-replete, iron-deficient, and iron-deficiency anaemia donor mice were 16.5±0.4, 11.5±0.4, and 7.0±1.4 [g/dL± ...
52nd ASH Annual …, 2010
... New York Presbyterian Hospital, NY 6 Pathology and Cell Biology, Columbia University, NewYork... more ... New York Presbyterian Hospital, NY 6 Pathology and Cell Biology, Columbia University, NewYork-Presbyterian Morgan Stanley Children&amp;amp;amp;amp;amp;amp;amp;#x27;s Hospital ... 2 * , Aggrey Dhabangi, MBChB, MMEd, (cand) 3 * , Deborah Nakiboneka-Ssenabulya, MBChB, MMEd 3 * , Henry Ddungu, MD 4 ...
…, 2005
Transfusion of red blood cells (RBCs) into patients with anti-donor RBC antibodies (crossmatch-in... more Transfusion of red blood cells (RBCs) into patients with anti-donor RBC antibodies (crossmatch-incompatible transfusion) can result in lethal antibody-mediated hemolysis. Less well appreciated is the ability of anti-RBC antibodies to specifically remove their target antigen from donor RBCs without compromising cell survival or adversely affecting the transfusion recipient. In an effort to elucidate the mechanistic details of this process, we describe the first animal model of nonhemolytic antibody-induced RBC antigen loss. RBCs from transgenic mHEL mice express surface hen egg lysozyme (HEL) as a transmembrane protein. Transfusion of mHEL RBCs into mice immunized with HEL results in selective loss of HEL antigen from donor RBCs without affecting other blood group antigens or reducing the circulatory life span of the transfused RBCs. While this process does not require the presence of a spleen, it requires both anti-RBC immunoglobulin G (IgG) antibodies and the Fc␥III receptor. These studies provide mechanistic insight into the phenomenon of antigen loss during incompatible transfusion in humans.
The insulin-dependent diabetic syndromes of the BB rat and man have several characteristics in co... more The insulin-dependent diabetic syndromes of the BB rat and man have several characteristics in common that implicate autoimmune processes in the etiology of pancreatic a cell destruction . The main arguments in favor of this are early infiltration of the islets by mononuclear cells (1, 2), and the presence of islet cell cytoplasmic (ICA)' and surface autoantibodies (ICSA) (3). ICSA are immunocytochemically detectable in the sera of patients with recent onset insulin-dependent diabetes mellitus (IDDM) and also in newly diagnosed diabetic BB rats . In vitro, ICSA can be cytotoxic for a cells (4-6), suggesting that these circulating autoantibodies may be direct effectors of a cell destruction in IDDM . However, what role they actually play in causing islet cell damage is still not completely clear. From this point of view it would be of interest to characterize (3 cell-specific autoantigens recognized by ICSA. Efforts have been made to isolate autoantigens using patient sera (7, 8) and mAbs against islets (9-11) . In this study we describe the successful preparation by hybridoma technology of a complement-mediated cytotoxic monoclonal autoantibody from a diabetic BB rat that reacts with rat insulinoma cells (RINm5F) and primary rat islet cells . The functional properties of this antibody and the antigens to which it binds are defined.
Vox Sanguinis, Jul 2, 2013
The hallmark of glucose-6-phosphate dehydrogenase (G6PD) deficiency is red blood cell (RBC) destr... more The hallmark of glucose-6-phosphate dehydrogenase (G6PD) deficiency is red blood cell (RBC) destruction in response to oxidative stress. Patients requiring RBC transfusions may simultaneously receive oxidative medications or have concurrent infections, both of which can induce haemolysis in G6PD-deficient RBCs. Although it is not routine practice to screen healthy blood donors for G6PD deficiency, case reports identified transfusion of G6PD-deficient RBCs as causing haemolysis and other adverse events. In addition, some patient populations may be more at risk for complications associated with transfusions of G6PD-deficient RBCs because they receive RBCs from donors who are more likely to have G6PD deficiency. This review discusses G6PD deficiency, its importance in transfusion medicine, changes in the RBC antioxidant system (of which G6PD is essential) during refrigerated storage and mechanisms of haemolysis. In addition, as yet unanswered questions that could be addressed by translational and clinical studies are identified and discussed.
EBioMedicine, Jan 16, 2016
Red blood cell (RBC) transfusions are essential for patients with hematological disorders and bon... more Red blood cell (RBC) transfusions are essential for patients with hematological disorders and bone marrow failure syndromes. Despite ABO matching, RBC transfusions can lead to production of alloantibodies against "minor" blood group antigens. Non-ABO alloimmunization is a leading cause of transfusion-associated mortality in the U.S. Despite its clinical importance, little is known about the immunological factors that promote alloimmunization. Prior studies indicate that inflammatory conditions place patients at higher risk for alloimmunization. Additionally, co-exposure to pro-inflammatory pathogen associated molecular patterns (PAMPs) promotes alloimmunization in animal models, suggesting that RBC alloimmunization depends on innate immune cell activation. However, the specific innate immune stimuli and sensors that induce a T cell-dependent alloantibody response to transfused RBCs have not been identified. The NLRP3 inflammasome senses chemically diverse PAMPs and damage ...
American Journal of Clinical Pathology, Jul 1, 1983
The neutralization of antibodies to the Lewis blood group systems is important in confirming the ... more The neutralization of antibodies to the Lewis blood group systems is important in confirming the presence of these antibodies and in investigating complex alloantibody problems. An improved method for neutralizing Lewis antibodies is described. Insoluble immunoadsorbents containing synthetic Lewis antigens are used to physically remove the antibody from serum. Thirty-five sera containing Lewis antibodies were neutralized completely using this technic; 23 non-Lewis alloantibodies were not inhibited. In contrast with current methods, this technic does not dilute the patient serum and results in an affinity purified serum sample.
Glycobiology, 2010
Alteration of glycoprotein glycans often changes various properties of the target glycoprotein an... more Alteration of glycoprotein glycans often changes various properties of the target glycoprotein and contributes to a wide variety of diseases. Here, we focused on the N-glycans of amyloid precursor protein whose cleaved fragment, beta-amyloid, is thought to cause much of the pathology of Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (AD). We previously determined the N-glycan structures of normal and mutant amyloid precursor proteins (the Swedish type and the London type). In comparison with normal amyloid precursor protein, mutant amyloid precursor proteins had higher contents of bisecting GlcNAc residues. Because N-acetylglucosaminyltransferase III (GnT-III) is the glycosyltransferase responsible for synthesizing a bisecting GlcNAc residue, the current report measured GnT-III mRNA expression levels in the brains of AD patients. Interestingly, GnT-III mRNA expression was increased in AD brains. Furthermore, beta-amyloid treatment increased GnT-III mRNA expression in Neuro2a mouse neuroblastoma cells. We then examined the influence of bisecting GlcNAc on the production of beta-amyloid. Both beta-amyloid 40 and beta-amyloid 42 were significantly decreased in GnT-III-transfected cells. When secretase activities were analyzed in GnT-III transfectant cells, alpha-secretase activity was increased. Taken together, these results suggest that upregulation of GnT-III in AD brains may represent an adaptive response to protect them from additional beta-amyloid production.
Blood, Nov 15, 2013
Because human immunodeficiency virus (HIV)-infected patients receive prophylaxis with oxidative d... more Because human immunodeficiency virus (HIV)-infected patients receive prophylaxis with oxidative drugs, those with glucose-6-phosphate dehydrogenase (G6PD) deficiency may experience hemolysis. However, G6PD deficiency has not been studied in the Dominican Republic, where many individuals have African ancestry. Our objective was to determine the prevalence of G6PD deficiency in Dominican HIV-infected patients and to attempt to develop a cost-effective algorithm for identifying such individuals. To this end, histories, chart reviews, and G6PD testing were performed for 238 consecutive HIV-infected adult clinic patients. The overall prevalence of G6PD deficiency (8.8%) was similar in males (9.3%) and females (8.5%), and higher in Haitians (18%) than Dominicans (6.4%; P = 0.01). By logistic regression, three clinical variables predicted G6PD status: maternal country of birth (P = 0.01) and a history of hemolysis (P = 0.01) or severe anemia (P = 0.03). Using these criteria, an algorithm was developed, in which a patient subset was identified that would benefit most from G6PD screening, yielding a sensitivity of 94.7% and a specificity of 97.2%, increasing the pretest probability (8.8-15.1%), and halving the number of patients needing testing. This algorithm may provide a cost-effective strategy for improving care in resource-limited settings.
Vox Sanguinis, Feb 1, 1983
Abstract. A solid-phase kinetic enzyme-linked immunosorbent assay has been developed to measure b... more Abstract. A solid-phase kinetic enzyme-linked immunosorbent assay has been developed to measure binding of antibodies to purified or synthetic blood group antigens and tested in the Lewis blood group system. Chemically synthesized Lewis a antigen is used as the target for the binding of serum antibody in this sandwich-type assay. Kinetic, rather than endpoint, determinations are used to calculate the amount of specific antibody. Data are presented showing the assay to be quantitative, sensitive, and specific. It can separately quantitate the amount of IgG or IgM anti-Lewis a present in patient sera. The assay uses commercially available reagents and is semiautomated. Thus, it will be useful for studies in quantitative immunohematology as other blood group antigens become available in purified form.
American Journal of Clinical Pathology, May 1, 2009
A curriculum in clinical pathology (CP) was developed under the auspices of the Academy of Clinic... more A curriculum in clinical pathology (CP) was developed under the auspices of the Academy of Clinical Laboratory Physicians and Scientists (ACLPS) in 2006. At the 2008 ACLPS meeting in Philadelphia, PA, a panel was convened to address the current challenges in resident education and how to overcome them. Current challenges include the heterogeneity of the discipline (which requires analytical, medical, and managerial knowledge), the diverse repertoire of clinical laboratory testing, and the need to better integrate the resident into the work flow of the laboratory, especially with respect to clinical consultation. Recommendations of the panel include the incorporation of active learning, clinical consultation, and competency assessment into CP resident training. A summary of the panel discussion is presented herein. by guest on
Transfusion, Apr 1, 2011
Retrospective studies suggest that the transfusion of older, stored red blood cells (RBCs) may be... more Retrospective studies suggest that the transfusion of older, stored red blood cells (RBCs) may be associated with increases in mortality, serious infections, multiorgan failure, thrombosis, and hospital length of stay. Our research is based on the overarching hypothesis that the adverse effects associated with transfusion of older, stored RBCs result from the acute delivery of hemoglobin iron to the monocyte-macrophage system. To test this "iron hypothesis," we are recruiting healthy human volunteers to donate double, leukoreduced, RBC units. We then transfuse them with one autologous fresh unit (i.e., after 3-7 days of storage) and one older, stored unit (i.e., at 40-42 days of storage). The primary study outcome will compare laboratory iron measures and proinflammatory cytokines after transfusion of fresh or older, stored RBCs. Similar studies using allogeneic RBC transfusions will be performed in chronically transfused patients with either sickle cell disease or b-thalassemia. Although prospective, randomized studies will ultimately determine the existence of adverse effects from transfusing older, stored RBCs, our goal is to determine the mechanism(s) for this potential effect.
The Journal of experimental medicine, Jan 16, 2016
Red blood cell (RBC) transfusion is a life-saving therapeutic tool. However, a major complication... more Red blood cell (RBC) transfusion is a life-saving therapeutic tool. However, a major complication in transfusion recipients is the generation of antibodies against non-ABO alloantigens on donor RBCs, potentially resulting in hemolysis and renal failure. Long-lived antibody responses typically require CD4(+) T cell help and, in murine transfusion models, alloimmunization requires a spleen. Yet, it is not known how RBC-derived antigens are presented to naive T cells in the spleen. We sought to answer whether splenic dendritic cells (DCs) were essential for T cell priming to RBC alloantigens. Transient deletion of conventional DCs at the time of transfusion or splenic DC preactivation before RBC transfusion abrogated T and B cell responses to allogeneic RBCs, even though transfused RBCs persisted in the circulation for weeks. Although all splenic DCs phagocytosed RBCs and activated RBC-specific CD4(+) T cells in vitro, only bridging channel 33D1(+) DCs were required for alloimmunizatio...
The Journal of Immunology, May 1, 1998
The M and N human blood group glycopeptide Ags are carried on RBCs by glycophorin A. Previous res... more The M and N human blood group glycopeptide Ags are carried on RBCs by glycophorin A. Previous results suggested that the murine humoral immune response against the N, but not the M, Ag is restricted. In addition, these results suggested that particular highly homologous heavy chains might be able to combine promiscuously with various light chains to yield anti-N specificity. To examine this, the current study used Fab phage methodology to couple an array of light chains, obtained from cDNA libraries isolated from immunized mice, to single Fd obtained from N61, N92, and 425/2B hybridomas. Interestingly, for the chimeric Fab to retain M or N specificity, the new light chains needed to belong to the same V k gene family as the light chain from the parental, hybridoma-derived mAb. In some cases the new light chains modified the Fab affinity and fine specificity. For example, libraryderived light chains coupled with the N92 Fd yielded chimeric Fab with increased affinity. In particular, the affinity of these univalent chimeric Fab for the N Ag was equivalent to that of the bivalent parental IgG mAb. Taken together, these results demonstrate that particular structures formed by the light chain V region are required to cooperate with a particular heavy chain V region to create a functional binding site for these glycopeptide Ags. They also demonstrate a lack of heavy chain promiscuity in the formation of murine anti-M and anti-N Abs.
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Papers by Steven Spitalnik