Douglas P. Blackall

Hemolytic uremic syndrome revisited: Shiga toxin, factor H, and fibrin generation.

The hemolytic uremic syndrome (HUS) is a disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. These features reflect the underlying histopathologic lesion: fibrin-rich thrombi that predominate in the renal microvasculature. HUS most commonly affects children younger than 5 years and is associated with Shiga toxin-producing enteric bacteria, the most important of which is Escherichia coli O157:H7. In this setting, HUS is epidemic and also might affect adults, particularly elderly people. Sporadic cases of HUS more commonly occur in adults and are associated with a wide variety of inciting agents and conditions. Although the disease manifestations might be similar and endothelial activation or injury likely represents a common etiologic event, differing responses to therapy suggest different pathogenic mechanisms. As more is understood about the underlying pathogenesis of the diseases that we now lump together as HUS, more efficacious and rational treatment and prevention strategies are likely to follow.

Comparison of RBCs and FFP with whole blood during liver transplant surgery

BACKGROUND : Component therapy has become the accepted standard of care in transfusion medicine. In instances of large blood loss, the transfusion of whole blood rather than the combination of RBCs and FFP is rational and may be preferred.

Clinical significance of anti-Jra: report of two cases and review of the literature.

BACKGROUND: Jra is a high‐frequency antigen seen in all populations, but the clinical significance of Jra antibodies is incompletely understood. Two cases are reported in which patients with anti‐Jra received incompatible transfusions.

Sialic acid-dependent binding of baculovirus-expressed recombinant antigens from Plasmodium falciparum EBA-175 to Glycophorin A.

The Plasmodium falciparum Erythrocyte Binding Antigen-175, EBA-175, is a soluble merozoite stage parasite protein which binds to glycophorin A surface receptors on human erythrocytes. We have expressed two conserved cysteine-rich regions, region II and region VI, of this protein as soluble His-tagged polypeptides in insect cell culture, and have tested their function in erythrocyte and glycophorin A binding assays. Recombinant region II polypeptides comprised of the F2 sub-domain or the entire region II (F1 and F2 sub-domains together) bound to erythrocytes and to purified glycophorin A in a manner similar to the binding of native P. falciparum EBA-175 to human red cells. Removal of sialic acid residues from the red cell surface totally abolished recombinant region II binding, while trypsin treatment of the erythrocyte surface reduced but did not eliminate recombinant region II binding. Synthetic peptides from three discontinuous regions of the F2 sub-domain of region II inhibited human erythrocyte cell binding and glycophorin A receptor recognition. Immune sera raised against EBA-175 recombinant proteins recognized native P. falciparum-derived EBA-175, and sera from malaria-immune adults recognized recombinant antigens attesting to both the antigenicity and immunogenicity of proteins. These results suggest that the functionally-active recombinant region II domain of EBA-175 may be an attractive candidate for inclusion in multi-component asexual blood stage vaccines.

Inhibition of erythroid progenitor cell growth by anti-Ge3.

The glycophorins C and D (GPC/D) are sialoglycoprotein components of the red cell membrane, which function to regulate shape and mechanical stability (Reid et al, 1987). Glycophorins carry the antigens of the Gerbich (Ge) blood group system including the high-incidence antigens Ge2, Ge3 and Ge4. Most infants born to women alloimmunised to Geantigens do not require treatment for haemolytic disease of the newborn (HDN). Generally, the infants have a positive directantiglobulin test, occasionally show mild jaundice, and do not require transfusion. However, two infants of one woman with anti-Ge3 had normal bilirubin values and reticulocytes at birth, but the neonatal outcomes were atypical, such that the infants developed late onset anaemia requiring transfusion a few weeks after birth (Arndt et al, 2005). Previous to these reports, similar unusual clinical features have been associated with anti-K HDN (Vaughan et al, 1994). Moreover, in vitro studies have demonstrated a suppression of erythropoiesis (Vaughan et al, 1998) and phagocytosis of early progenitor cells (Daniels et al, 2003). Arndt et al (2005) demonstrated in vitro macrophage-mediated phagocytosis of early erythroid progenitor cells by anti-Ge3. Here, we report on the inhibitory effect of anti-Ge3 on in vitro erythroid progenitor cell growth. Two examples of maternal anti-Ge3 associated with HDN were evaluated; one with a titre of 256 (Arndt et al, 2005) and the other with a titre of 32, together with a murine monoclonal anti-GPC. Cord blood mononuclear cells (1Æ5 · 10) were cultured in MethoCult media (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 3 U/ml of erythropoietin. Cultures were established in the presence of 20% (v/v) maternal sera, Iscove’s modified Dulbecco’s medium (IMDM) containing 10 lg/ml of murine immunoglobulin (Ig) G1 monoclonal anti-GPC (Abcam Ltd, Cambridge, UK), or an irrelevant murine IgG1 in IMDM as a control (Serotec, Raleigh, NC, USA). Polyclonal anti-K (Dominion Biological Limited, Dartmouth, NS, Canada) in IMDM and maternal anti-D serum (titre 256) served as positive and negative controls respectively. MethoCult media containing erythropoietin was used to determine the maximum colony growth. Sera were evaluated in triplicate, and the number of erythroid colony-forming units (CFU-E) and burst-forming units (BFUE) were evaluated after 7 and 14 d of incubation at 37 C/5% CO2 respectively. The statistical significance for the mean number of colonies per well compared with culture media without antibodies was evaluated using a two-sided Student’s t-test, with P < 0Æ05 considered to be significant. Both maternal sera containing anti-Ge3, the murine antiglycophorin C and anti-K significantly inhibited BFU-E and CFU-E growth (Fig 1). The maternal anti-D and the murine

Warm-reactive autoantibodies in pediatric patients: clinical and serologic correlations.

The significance of warm-reactive autoantibodies in pediatric patients has not been a subject of thorough evaluation. This study was undertaken to correlate the clinical and serologic features of these antibodies to identify predictors of clinical significance. Forty-two consecutive patients with serologically detectable warm-reactive autoantibodies were studied. These patients (21 male, 21 female) had a mean age of 9 years (range: 2 mo to 21 y). Primary diagnoses included autoimmune disorders (14), sickle cell disease (14), viral infection (4), idiopathic autoimmune hemolytic anemia (2), leukemia (2), and other diseases (6). Autoimmune hemolysis, as determined by clinical and laboratory findings, was documented in 24 patients (57%). Serologic studies revealed that all patients demonstrated IgG on their red cells [Direct Antiglobulin Test (DAT) reactivity range: microscopic to 3+]; 17 (40%) also demonstrated complement (DAT reactivity range: microscopic to 2+). There was a correlation between the strength of the DAT for IgG and the presence of complement on the red cells, with both being important predictors of hemolysis. These findings may be useful in predicting the clinical significance of warm-reactive autoantibodies in pediatric patients and allow for more efficient and effective follow-up care.

Antibiotic hypersensitivity in CF: Drug-induced life-threatening hemolytic anemia in a pediatric patient

Adverse reactions to antibiotics in patients with cystic fibrosis (CF) are a growing concern. We report the case of a pediatric patient with CF with multiple comorbidities and a history of drug reactions, who developed life-threatening piperacillin-induced immune hemolytic anemia. We review drug-induced hemolytic anemia (DIIHA) in particular, and antibiotic hypersensitivity in CF in general, including the frequency, pathogenesis, and risk factors. Finally, we discuss the treatment options and propose an algorithm for the management of drug-induced hypersensitivity reactions in patients with CF.

Cryoprecipitate-reduced plasma:rationale for use and efficacy in the treatment ofthrombotic thrombocytopenic purpura

840 TRANSFUSION Volume 41, June 2001 www.transfusion.org Cryoprecipitate-reduced plasma (CRP), also termed cryopoor plasma, cryoprecipitate-poor plasma, or cryosupernatant, is that fraction of plasma remaining after the removal of cryoprecipitable proteins (i.e., cryoprecipitate). As such, it is plasma from which approximately one-half of the fibrinogen, factor VIII, and fibronectin have been removed.1 CRP is also depleted of the largest multimers of vWF.1 This plasma fraction, because of the blood components it lacks, can no longer be used as a source of FFP. Instead, it is routinely used as a raw material in manufacturing albumin solutions, therapeutic immunoglobulins, and non-FVIII coagulation factor concentrates. More recently, however, CRP has been used in the treatment of thrombotic thrombocytopenic purpura (TTP), either initially or after a trial of FFP.2-7 This review will focus on the use of CRP as a treatment for TTP, the only disease for which this plasma fraction is specifically used as a therapeutic modality.

Hemolytic Disease of the Fetus and Newborn Due to Anti-Ge3: Combined Antibody-Dependent Hemolysis and Erythroid Precursor Cell Growth Inhibition

The Gerbich (Ge) antigens are a collection of high-incidence antigens carried on the red blood cell membrane glycoproteins, glycophorins C and D. Antibodies against these antigens are uncommon, and there have been only rare case reports of hemolytic disease of the fetus and newborn due to anti-Ge. In this case report, we present a neonate with severe anemia and hyperbilirubinemia due to anti-Ge3. Routine and special laboratory studies undertaken in this case suggested two mechanisms for the patients hemolysis and persistent anemia. Antibody-dependent hemolysis was associated with early-onset hyperbilirubinemia, anemia, and a mild reticulocytosis, and inhibition of erythroid progenitor cell growth was associated with late anemia and normal bilirubin and reticulocyte values. Though rare, anti-Ge3 can be a dangerous antibody in pregnancy. Affected neonates may require intensive initial therapy and close follow-up for at least several weeks after delivery.

Recombinant polymeric IgG anti-Rh: a novel strategy for development of direct agglutinating reagents.

Anti-Rh alloantibodies are used in research and clinic laboratories to define the Rh antigenic profile of human blood samples. IgM anti-Rh antibodies directly agglutinate Rh-positive RBCs. Anti-Rh antibodies of the IgG isotype bind to Rh antigens with a higher intrinsic affinity than IgM and sensitize RBCs, but do not induce direct hemagglutination. The aim of this work was to produce IgG anti-Rh possessing direct hemagglutinating properties of IgM. To achieve this goal, recombinant antibody technology was used to construct genes encoding Ig light and heavy chains that will form polymers with anti-Rh specificity. Expression vectors and liposome-mediated DNA transfer were used to generate transfectomas secreting human recombinant IgG3 anti-Rh. ELISA, SDS-PAGE, and hemagglutination were used to identify and characterize the recombinant antibody produced. Thus, a recombinant polymeric IgM-like IgG3 anti-Rh antibody was produced that directly agglutinates RBCs with specificity identical to that of the parent non-agglutinating IgG. The results obtained suggest that the technology used here to generate polymeric IgM-like IgG3 anti-Rh antibodies can be applied to produce Rh blood typing reagents. This approach might also be used to develop reagents for which cell surface antigen binding and agglutination or aggregation is required.