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Autoimmune lymphoproliferative syndrome (ALPS), is an inherited disorder characterized by defective lymphocyte apoptosis, lymphadenopathy, splenomegaly, accumulation of T-cell receptor (TCR)-αβrscript>+ CD4− CD8− T cells (double-negative T cells) and autoimmunity. We investigated the incidence and nature of neutrophil and platelet antibodies in patients with ALPS. Sera from 26 patients with ALPS were tested for neutrophil antibodies by granulocyte immunofluorescence, granulocyte agglutination and monoclonal antibody immobilization assays of granulocyte antigens, and for platelet antibodies using a solid-phase antibody-detection system. Neutrophil antibodies were detected in 46% of patients with ALPS. Antibody specificity could be defined in eight of the 12 patients with neutrophil antibodies. Among these eight patients, four had antibodies directed against more than one antigen. Overall, 14 antibodies directed to specific antigens were identified: three were directed to the HNA-1a antigen of FcγRIIIb; two to the HNA-1b antigen of Fcγ-RIIIb; two to epitopes common to all FcγRIIIb molecules; four to the HNA-2a antigen of the NB1 glycoprotein; and three to neutrophil β2 integrins. Platelet antibodies were detected in 35% of patients with ALPS. No antibody specificities were identified among the platelet antibodies. There was no association between the detection of neutrophil antibodies and a history of clinical neutropenia, or between the detection of platelet antibodies and a history of clinical thromobocytopenia. Neutrophil and platelet antibodies are important markers of ALPS, but do not always cause clinical cytopenias. The specificities of neutrophil antibody were similar to those found in children with autoimmune neutropenia but without ALPS. [ABSTRACT FROM AUTHOR] more...

Examines the ontogeny of NBI antigen expression by hematopoietic progenitor cells. Examination of ontogeny and frequency of granulocytic cells; Similarity on the size of NBI-positive neurophil subpopulation among adults and children; Comparison between the percentage of myelocyte and the frequency of NBI-positive neurophil. more...

Background: Severe malarial anemia is a leading cause of death in African children under 3 years of age who are infected by Plasmodium(P)falciparum. Pathogenesis of this anemia is not understood. It has been hypothesized that it may be due to the effects of the parasite on host's red blood cells (RBCs), the immune system's battle to fight off the parasite, or dyserythropoiesis. Whether induced by a single or multiple factors, uninfected as well as infected RBCs are rapidly cleared from circulation. The purpose of this study was to determine if P falciparum induces changes in RBC membranes that contributed to the immune destruction of RBCs. Study Design and Methods: RBCs were collected from healthy subjects and tested using standard hemagglutination assays for 45 antigens representing 21 blood group systems and antigen collections before and after exposure to P falciparum, strain FVO. Lectins were used to determine whether crypt or neoantigens were expressed on the RBC membrane. Polybrene was used to detect changes in sialic acid. RBCs were cultured in vitro with and without the parasite and blinded serologic studies were completed. CD35(Complement Receptor 1), CD55(Decay Accelerating Factor), CD59(Membrane Inhibitor of Reactive Lysis) and CD47(Integrin-associated protein) expression was assessed by flow cytometry and compared for infected and uninfected RBCs. Percent parasitemia was determined using Giemsa stained thin blood films. Results: Two (Ch, Lu[sup b]) of 45 antigens tested had differing strengths of agglutination between infected and uninfected RBCs but were resolved with a second source of antisera. 43 antigens showed no significant differences in strength of agglutination between the infected and uninfected RBCs. Lectin and polybrene testing showed no differences. CD35, CD55, CD59 and CD47 levels showed no statistically significant differences. Conclusion: P falciparum does not appear to alter the expression of classified immunogenic antigens on the RBC membrane in this in vitro system. Previously observed changes in CD35, CD55, and CD59 levels on RBCs of infected hosts that were not observed in this study indicate that these protein levels may be controlled by the host immune system. Perhaps evaluating CD47 levels in infected hosts will provide insight into the cause of anemia. Pathogenesis of the hemolytic episode that occurs in these children remains unclear but would appear to be immune mediated. [ABSTRACT FROM AUTHOR] more...

Background: Granulocyte donors are frequently given granulocyte colony-stimulating factor (G-CSF) with or without dexamethasone (Dex) approximately 18 hours prior to apheresis to increase granulocyte (gran) counts and yields. We assessed the kinetics of gran mobilization during the first 8 hours after G-CSF administration to determine if grans can be mobilized and collected on the same day. Study Design: Twelve subjects underwent four mobilization cycles each. In random order, they received either subcutaneous (SQ) GCSF 5 µg/kg, intravenous (IV) G-CSF 5 µg/kg, SQ G-CSF 5 µg/kg plus oral Dex 8 mg, or IV G-CSF 5 µg/kg plus oral Dex 8 mg. Gran counts were measured prior to and at ½, 1, 2, 4, 6, 8 and 24 hours following drug administration. Results: Following all four mobilization regimens, mean gran counts fell markedly one half hour after the mobilizing agents were given, rose above baseline levels at 2 hours, and increased further with each time interval up to 8 hours. In the absence of Dex, at 4, 6, and 8 hours after GCSF, there was no difference in gran counts between the SQ or IV G-CSF administration routes. The addition of Dex significantly increased the mobilization response to both SQ and IV G-CSF from hours 4 through 24. Through hour 8, there was no difference in the degree of gran mobilization among the SQ G-CSF plus Dex and IV G-CSF plus Dex regimens. However, at 24 hours, gran counts were sustained at a higher level with SQ G-CSF plus Dex than with IV G-CSF plus Dex. Conclusions: The gran mobilization response to SQ G-CSF plus Dex is sustained at peak levels from 8 to 24 hours after co-administration of the two drugs, allowing optimal granulocytapheresis yields to be obtained as early as 8 hours (same-day) after drug administration. There is no advantage to giving G-CSF by the IV route. [ABSTRACT FROM AUTHOR] more...

Background: Gel microcolumns are used to detect unexpected serum antibodies and to determine ABO blood group and Rh D type. Direct antiglobulin tests (DATs) also are performed with this system. The purpose of this study was to determine if gel microcolumn anti-IgG cards can be used to detect antibodies eluated from red blood cells (RBCs). Study design: DATs were performed on all samples by tube agglutination and gel microcolumn assays. Acid eluates were prepared from DAT-positive RBCs from 17 peripheral blood and 35 umbilical cord blood samples. Specificity of eluates was determined by both tube and gel assays using three screening cells plus A1 and B cells, when deemed appropriate. The cord blood samples were from group A or B neonates suspected of having ABO hemolytic disease of the newborn. The peripheral blood samples were from patients with autoimmune hemolytic anemia (AIHA), who had a suspected transfusion reaction, and who were recipients of ABO-mismatched hematopoietic stem cell transplants. As controls, eluates were prepared and tested from DAT-negative RBCs from 6 umbilical cords and 6 healthy donors. Eluates were also prepared and screened for antibody specificity from samples that had been previously tested and found to have positive DATs and negative eluates by tube testing. Results: All 17 peripheral blood samples had positive DATs when tested by gel assays and 15 had positive DATs by tube assays. Eluates were prepared and 10 of 17 eluates were positive in both assays, 5 were negative in both assays, and 2 from patients with AIHA were positive in gel assays and negative in tube assays. All 35 umbilical cord blood samples had positive DATs in gel assays and 34 were positive in tube assays. Thirty-three of the 35 eluates were positive in both assays. Two of the 35 eluates reacted with both A1 and B cells in tube assays, but no reactivity was seen in gel assays. In two of the 33 eluates that were positive in both assays, the antibody reactivity differed between the two assays. When tested in tube assays, the 2 eluates reactive with both A1 and B cells. When the same eluates were tested in gel assays, one reacted only with A1 cells and the other only with B cells. The 7 samples with positive-DATs but negative eluates as determined by tube assays had negative gel DATs and negative eluates. Negative tube and gel DATs were observed in the 12 control samples and eluates prepared were negative in both assays. Conclusions: Results of testing eluates in gel assays were similar to those obtained in tube assays. Gel assays may be better at detecting antibodies eluated from RBCs from patients with AIHA and tube assays better at detecting isoagglutinins eluted from umbilical cord RBCs. [ABSTRACT FROM AUTHOR] more...

Background: Alloimmunization to the human neutrophil alloantigen HNA-2a (NB1) plays an important part in alloimmune neonatal neutropenia, immune neutropenia after bone marrow transplantation, drug-induced immune neutropenia and in transfusion-related acute lung injury (TRALI). The NB1 glycoprotein, which has been recently clustered as CD177, is expressed on the neutrophil plasma membrane of 88% to 97% of healthy individuals. In this study, we elucidated the primary structure of the NB1 glycoprotein and the molecular basis of the HNA-2a (NB1)-negative phenotype. Methods: By the use of the well-characterized HNA-2a-specific monoclonal antibody 7D8, we isolated the antigen by immunoaffinity chromatography. The molecular mass was determined by MALDI-TOF mass spectrometry. After elucidation of the N-terminal amino acid sequence by Edman degradation primers we designed primers for rapid amplification of cDNA ends (RACE) PCR. The obtained PCR products were sequenced and COS-7 cells were transfected with the obtained cDNA. In addition, we investigated the neutrophils of two individuals who did not express the HNA-2a (NB1) antigen on their neutrophils. Results: The Mr of the NB1 glycoprotein was found to be 50.6 kDa with a 7.5 kDa carbohydrate moiety. In the obtained 1,614 bp cDNA, a 1,311 bp sequence was identified to represent the entire coding region. Transfected COS-7 cells expressed the HNA-2a (NB1) antigen on their membrane. The DNA sequence encoding a protein of 416 amino acids and a 21 amino acid signal peptide was found to be spread over nine exons in the genome. The deduced amino acid sequence predicted a protein with 3 N-linked carbohydrate binding sites, 2 cysteine-rich domains, and a glycosylphosphatidylinositol (GPI)-attachment (v) site. Homology searches indicated that the NB1 glycoprotein belongs to the urokinase-type plasminogen activator receptor (uPAR)/CD59/Ly-6 or uPAR superfamily. In the genome of two HNA-2a negative individuals, we identified the HNA-2a coding sequence. However, NBl-related mRNAs of the two individuals always showed accessory sequences which were considered to be introns because of the typical acceptor and donator splicing sites. Conclusions: We characterized the neutrophil-specific HNA-2a (NB1) alloantigen as a GPI-anchored 50.6 kDa glycoprotein which belongs to the uPAR/CD59/Ly-6 (uPAR) superfamily. The HNA-2a (NB1)-negative phenotype is most likely the result of an expression defect and not of a gene deletion. [ABSTRACT FROM AUTHOR] more...