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Red blood cell (RBC) antibodies can persist for decades or decrease quickly to undetectable levels. Antibody persistence has not been systematically studied. Women whose children are treated with intrauterine transfusions (IUT) for haemolytic disease of the fetus (HDFN) often produce additional antibodies, which can be evoked by the intrauterine transfusion or by fetomaternal haemorrhage during the procedure. Factors associated with persistence of both the antibodies responsible for HDFN and additional antibodies were studied in 260 women whose children were treated with IUT between 1988 and 2008. They possessed 499 (205 anti-D and 294 non-D) antibodies after the last IUT. After a median follow-up of 8·7 years, all 260 antibodies primarily responsible for HDFN had persisted. Additional antibodies directed against antigens of the children persisted in 70·6%, and in 32·3% if they were not child-specific (P < 0·001). Antibodies induced by irradiated IUT persisted in only 7·1%. Multivariate analyses showed that non-HDFN antibody persistence was dependent on the antibody titre and specificity. In conclusion, persistence of antibodies mainly depends on antibody strength and specificity. Difference between fetal or non-fetal immunogens suggests maintenance of antigenic stimulation possibly by long-term fetomaternal chimerism., (© 2014 John Wiley & Sons Ltd.)

In a prospective study of 109 subjects, an enzyme-linked immunosorbent assay (ELISA) was used to measure platelet-rich plasma (PRP) serotonin levels in patients with polycythaemia vera (PV; n = 27), essential thrombocythaemia (ET; n = 14), myelofibrosis with myeloid metaplasia (MMM; n = 30), secondary or spurious polycythaemia (SP; n = 22) and controls (n = 16). Nine study subjects who were taking a selective serotonin reuptake inhibitor (SSRI) all displayed a markedly decreased PRP serotonin level (median, 24.2 ng/10(9) platelets; range, 0-49.3) and were therefore excluded from further analysis. Among the remaining 100 subjects, the median and range of PRP serotonin levels, in ng/10(9) platelets, was significantly lower in MMM (89.5; 0-278.3), PV (204.8; 0-496.0) and ET (385.3; 136.8-1025.7) compared with both SP (608.8; 369.0-1780.1) and controls (567.2; 359.9-1071.1). Neutrophil polycythaemia rubra vera-1 (PRV-1) expression was concurrently assayed by real-time polymerase chain reaction in 69 patients (23 PV, 17 SP, 12 ET, seven MMM, 10 controls). PRP serotonin measurement performed as well as the PRV-1 assay in distinguishing PV from SP (93% vs. 86% test accuracy). The current study suggests that PRP serotonin concentration might be considered as one of the several biological markers that complement each other for the diagnosis of PV., (Copyright 2004 Blackwell Publishing Ltd)

The aims of this study were to determine whether the severity of fetomaternal alloimmune thrombocytopenia (FMAIT) in the current pregnancy could be predicted from the history of FMAIT in previous pregnancies, and to assess the effects of different types of antenatal intervention. Fifty-six fetuses were studied that all had a sibling affected by FMAIT due to human platelet antigen 1a (HPA-1a) alloimmunization. Cases with a sibling history of antenatal intracranial haemorrhage (ICH) or severe thrombocytopenia (platelet counts of < 20 x 109/l) had significantly lower pretreatment platelet counts than cases whose siblings had less severe thrombocytopenia or postnatal ICH. Maternal therapy resulted in a platelet count exceeding 50 x 109/l in 67% of cases. None of the fetuses managed by serial platelet intrauterine transfusions (IUT) suffered ICH following treatment. However, several serious complications arose with fetal blood sampling (FBS). Overall, intervention improved outcome, as three study cases suffered from antenatal ICH and three others died whereas 15 study cases had a sibling with an ICH, eight of whom died. The results of this study suggest that the start of therapy can be stratified on the basis of the sibling history of FMAIT, and support the use of maternal therapy as first-line treatment.

We previously reported the initial characterization of five human single-chain Fv (scFv) antibody fragments specific for the blood group antigens B, D(Rh), E(Rh), Kpb and HI. The scFvs were isolated from a phage-antibody library constructed from the variable region genes of two non-immunized donors. In this paper we report the specificity, affinity and kinetics of antigen binding of these scFv fragments. All five scFvs agglutinated the appropriate red cell phenotype following the addition of a monoclonal antibody which recognizes a peptide tag incorporated into the scFv. The anti-B and anti-HI scFv molecules, which recognize high density carbohydrate antigens, spontaneously polymerized and agglutinated red cells directly. None of the antibody fragments showed cross-reactivity with other red cell antigens, with the exception of the anti-E which reacted weakly with E-negative cells. Specific scFv binding was confirmed by ELISA, flow cytometry and radioactive labelling. The anti-D scFv recognized 17,600 sites on cDE/cDE red cells with an association constant (Ka), of 5.2 x 10(7) M-1 and a rate constant for dissociation (koff) of 1.9 x 10(-2) s-1. The anti-E scFv recognized 29,800 and 39,800 sites on cDE/cDE red cells in two experiments with Kas of 8.4 x 10(6) and 4.4 x 10(7) M-1. The koff for this antibody was 2.7 x 10(-2) s-1. The results demonstrate that scFv antibody fragments specific for cell surface antigens and possessing affinities typical of the primary immune response can be obtained from a phage-display library.

An RNA-based method has been developed to genotype donors for the granulocyte-specific alloantigens NA1 and NA2. mRNA was isolated from granulocytes, reversely transcribed into cDNA and amplified using an Fc-gamma-receptor III-1 sequence-specific primer in the polymerase chain reaction (PCR). PCR products were analysed by restriction fragment length polymorphism (RFLP) using the restriction endonuclease Taq I, which provided a distinct restriction fragment pattern corresponding to the NA alleles. 17 donors were typed by PCR-RFLP and the results were in close accordance with those obtained by serological phenotyping by granulocyte immunofluorescence and the antigen capture assay MAIGA.

The human neutrophil-specific alloantigen NB1 was identified as a glycosyl-phosphatidylinositol (GPI)-anchored N-glycosylated protein of M(r) 56-62 kD under reducing conditions. Under non-reducing conditions its M(r) was 49-55 kD. This glycoprotein antigen was found to be expressed by only a subpopulation of normal donor neutrophils, and could not be detected on other blood cells. The allotypic epitope recognized by human anti-NB1 IgG was also recognized by the mouse monoclonal antibody 1B5. The percentage of neutrophils stained by these antibodies varied greatly among healthy donors (range 0-100%). When 16 donors were repeatedly tested, the NB1-positive neutrophil fraction appeared to remain remarkably constant over time in most donors, but significant fluctuations were seen in some. NB1 antigen was found to be expressed not only on the plasma membrane, but also intracellularly on the membranes of small vesicles and specific granules. The neutrophils which expressed NB1 antigen on the plasma membrane were the same as those with intracellular expression of this antigen. Crosslinking of NB1 antigen on the plasma membrane with monoclonal antibody 1B5 and goat-anti-mouse Ig resulted in internalization of the complex, while in-vitro stimulation of neutrophils caused an increase of the intensity of plasma membrane staining with anti-NB1, but only of those cells that were positive already prior to stimulation. The NB1 glycoprotein thus appears to identify a distinct subset of neutrophils, the size of which greatly varies among healthy donors. The function of the NB1 glycoprotein remains unclear, but its behaviour upon crosslinking and chemotactic peptide stimulation suggests a possible role as receptor molecule.