Characterization and quantification of suspended sediment sources to the Manawatu River, New Zealand.

Knowledge of sediment movement throughout a catchment environment is essential due to its influence on the character and form of our landscape relating to agricultural productivity and ecological health. Sediment fingerprinting is a well-used tool for evaluating sediment sources within a fluvial catchment but still faces areas of uncertainty for applications to large catchments that have a complex arrangement of sources. Sediment fingerprinting was applied to the Manawatu River Catchment to differentiate 8 geological and geomorphological sources. The source categories were Mudstone, Hill Subsurface, Hill Surface, Channel Bank, Mountain Range, Gravel Terrace, Loess and Limestone. Geochemical analysis was conducted using XRF and LA-ICP-MS. Geochemical concentrations were analysed using Discriminant Function Analysis and sediment un-mixing models. Two mixing models were used in conjunction with GRG non-linear and Evolutionary optimization methods for comparison. Discriminant Function Analysis required 16 variables to correctly classify 92.6% of sediment sources. Geological explanations were achieved for some of the variables selected, although there is a need for mineralogical information to confirm causes for the geochemical signatures. Consistent source estimates were achieved between models with optimization techniques providing globally optimal solutions for sediment quantification. Sediment sources was attributed primarily to Mudstone, ≈38-46%; followed by the Mountain Range, ≈15-18%; Hill Surface, ≈12-16%; Hill Subsurface, ≈9-11%; Loess, ≈9-15%; Gravel Terrace, ≈0-4%; Channel Bank, ≈0-5%; and Limestone, ≈0%. Sediment source apportionment fits with the conceptual understanding of the catchment which has recognized soft sedimentary mudstone to be highly susceptible to erosion. Inference of the processes responsible for sediment generation can be made for processes where there is a clear relationship with the geomorphology, but is problematic for processes which occur within multiple terrains.

Neutrophil and platelet antibodies in autoimmune lymphoproliferative syndrome.

BACKGROUND AND OBJECTIVES: Autoimmune lymphoproliferative syndrome (ALPS), is an inherited disorder characterized by defective lymphocyte apoptosis, lymphadenopathy, splenomegaly, accumulation of T-cell receptor (TCR)-alphabeta+ 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. MATERIALS AND METHODS: 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. RESULTS: 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 FcgammaRIIIb; two to the HNA-1b antigen of Fcgamma-RIIIb; two to epitopes common to all FcgammaRIIIb molecules; four to the HNA-2a antigen of the NB1 glycoprotein; and three to neutrophil beta2 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. CONCLUSIONS: 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.

A preliminary comparison of flow cytometry and tube agglutination assays in detecting red blood cell-associated C3d.

This study compared flow cytometric analysis with tube agglutination assays for the detection of red blood cell (RBC)-associated complement and immunoglobulins (Igs). RBCs from 20 patients with reactive tube direct antiglobulin tests (DATs) were evaluated by flow cytometry with anti-C3d, anti-IgG, anti-IgM and anti-IgA. Serial samples were also tested from a patient at risk of passenger lymphocyte haemolysis. Results of flow cytometry and tube assays for anti-IgG were as follows: 12 of 20 samples reactive in both; six of 20 nonreactive in both; two of 20 discordant with a reactive tube and a nonreactive flow cytometry assay. Anti-C3d results showed nine of 20 reactive in both and 11 of 20 discordant with a nonreactive tube and a reactive flow cytometry assay. In the IgM flow cytometry assay, three samples were reactive with anti-IgM. Samples from a group A woman who was transplanted with stem cells from a group B donor showed that on days 3 through 6 post-transplant, the flow cytometry assays for anti-IgG and/or anti-C3d were reactive, whilst the tube assays were nonreactive. In conclusion, flow cytometric analysis is more sensitive than the tube assay for the detection of RBC-associated C3d. Further studies are needed to determine the correlation of C3d levels with clinical sequelae.

A novel method using formamide for the elution of antibodies from erythrocytes.

BACKGROUND AND OBJECTIVES: Accurate identification of antibodies that sensitize red blood cells (RBCs) involves dissociating them from RBCs using an in vitro elution method that does not alter their antigen-binding properties, and analysis of the eluates against a panel of RBCs. MATERIALS AND METHODS: A method was developed that allowed efficient RBC antibody elution. Human polyclonal anti-D was used to sensitize Rh-positive RBCs, and known antigen-antibody disruptive reagents were tested using these RBCs. The best reagent conditions were optimized. Eluates made were tested and compared to results obtained with a glycine-acid-based commercial elution kit to determine efficacy. Patient samples that were positive with direct antiglobulin tests (DATs), and in vitro commercial antisera-sensitized RBCs representing clinically significant antibodies, were used for evaluating the new method. RESULTS: The formamide method was efficient at removing antibodies from RBCs. The patient samples with a positive DAT had antibodies recovered with the same specificity when compared to the acid-based technique. The length of preparation time was similar for both formamide and acid-based methods. Results of testing the eluates made from reagent RBCs sensitized with commercial antisera were distinct with antigen-positive and -negative erythrocytes. CONCLUSIONS: The formamide method compares well with acid techniques and may be an alternative choice of elution method.

In vitro RBC exposure to Plasmodium falciparum has no effect on RBC antigen expression.

Severe malarial anaemia is a leading cause of death in African children younger than 3 years of age who are infected with Plasmodium falciparum. The pathogenesis of this anaemia is not understood. The purpose of this study was to determine if P. falciparum induces changes in RBC membranes that contribute to the immune destruction of RBCs. RBCs were collected from healthy subjects and tested using standard haemagglutination assays for 45 antigens representing 21 blood group systems/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) flow cytometric assays were compared for infected and uninfected RBCs. The percentage of parasitaemia was determined using Giemsa-stained thin blood films. Two (Ch, Lub) of the 45 antigens had differing strengths of agglutination between infected and uninfected RBCs, but these differences were resolved with a second source of antisera. Forty-three antigens showed no significant differences in the strength of agglutination between the infected and uninfected RBCs. Lectin and polybrene testing showed no differences. CD35, CD55, CD59 and CD47 levels showed no significant differences. P. falciparum does not appear to alter the expression of classified immunogenic antigens on the RBC membrane in this in vitro system. The pathogenesis of the haemolytic episode that occurs in these children remains unclear.

Comparison of DATs using traditional tube agglutination to gel column and affinity column procedures.

BACKGROUND: Detection of immunoglobulin or complement bound to RBCs by using the DAT is valuable in the diagnosis of immune-mediated hemolytic anemia. Traditionally, the DAT has been performed by tube agglutination using anti-IgG or anti-C3d. The purpose of this study was to compare the tube agglutination DAT to gel microcolumn, affinity microcolumn, and flow cytometric DATs on RBCs coated in vitro and on patient RBC samples. STUDY DESIGN AND METHODS: RBCs from 84 patients were assessed by tube agglutination DAT, one gel microcolumn DAT, and two affinity microcolumn DATs. One affinity microcolumn assay was unmodified and one was modified by the addition of polyspecific antiglobulin or anti-IgG as a secondary antibody. RBCs from 15 of the 84 patients underwent analysis by flow cytometry with fluorescence-labeled anti-IgG. The assays were also compared by using D+ RBCs sensitized with serially adjusted concentrations of anti-D. RESULTS: Both tube agglutination and gel microcolumn DATs were positive in 49 patient samples; both assays were negative in 20 samples, and the results were discordant in 15. Gel microcolumn DATs were more likely than were tube agglutination DATs to detect IgG on RBCs. Affinity microcolumn DATs were less likely than gel microcolumn or tube agglutination DATs to detect IgG on RBCs. Flow cytometry results were the same as gel microcolumn results in 12 of 15 patient samples and the same as tube agglutination results in 13 of 15. Tube agglutination and both affinity microcolumn assays reacted with RBCs coated with anti-D that was diluted 1-in-100. The gel microcolumn and flow cytometry assays reacted with RBCs coated with anti-D diluted 1-in-400. There was no correlation between tube agglutination and gel microcolumn DATs in detecting bound C3d. CONCLUSION: Detection of IgG bound to RBCs was not consistent with the methods described. Gel microcolumn DATs were more sensitive than tube agglutination and affinity microcolumn DATs. Given the varied results of these assays, reference laboratories should not rely on a single method for DATs. More comprehensive testing should be performed when the tube agglutination DAT is negative in a patient with suspected immune-mediated hemolytic anemia. Further comparisons are necessary to determine the proficiency of flow cytometric assays.

Delayed donor red cell chimerism and pure red cell aplasia following major ABO-incompatible nonmyeloablative hematopoietic stem cell transplantation.

Delayed donor red cell engraftment and pure red cell aplasia (PRCA) are well-recognized complications of major ABO-incompatible hematopoietic stem cell transplantation (SCT) performed by means of myeloablative conditioning. To evaluate these events following reduced-intensity nonmyeloablative SCT (NST), consecutive series of patients with major ABO incompatibility undergoing either NST (fludarabine/cyclophosphamide conditioning) or myeloablative SCT (cyclophosphamide/high-dose total body irradiation) were compared. Donor red blood cell (RBC) chimerism (initial detection of donor RBCs in peripheral blood) was markedly delayed following NST versus myeloablative SCT (median, 114 versus 40 days; P <.0001) and strongly correlated with decreasing host antidonor isohemagglutinin levels. Antidonor isohemagglutinins declined to clinically insignificant levels more slowly following NST than myeloablative SCT (median, 83 versus 44 days; P =.03). Donor RBC chimerism was delayed more than 100 days in 9 of 14 (64%) and PRCA occurred in 4 of 14 (29%) patients following NST, while neither event occurred in 12 patients following myeloablative SCT. Conversion to full donor myeloid chimerism following NST occurred significantly sooner in cases with, compared with cases without, PRCA (30 versus 98 days; P =.008). Cyclosporine withdrawal appeared to induce graft-mediated immune effects against recipient isohemagglutinin-producing cells, resulting in decreased antidonor isohemagglutinin levels and resolution of PRCA following NST. These data indicate that significantly delayed donor erythropoiesis is (1) common following major ABO-incompatible NST and (2) associated with prolonged persistence of host antidonor isohemagglutinins. The clinical manifestations of these events are affected by the degree and duration of residual host hematopoiesis.

The expression of NA antigens in people with unusual Fcgamma receptor III genotypes.

BACKGROUND: The Fcgamma receptor IIIb (FcgammaRIIIB) genes that encode neutrophil-specific antigens NA1 and NA2 differ at 5 nucleotides (nts); in 4, the result is an amino acid (AA) difference between the two alleles. The role of each of these differences in antigen expression is not known. Persons with FcgammaRIIIB genes that differ from NA1-FcgammaRIIIB and NA2-FcgammaRIIIB by 1 nt have been described. This study compared NA1 and NA2 expression on granulocytes in persons with variant FcgammaRIIIB genes and in healthy blood donors. STUDY DESIGN AND METHODS: Reactions of NA1- and NA2-specific MoAbs and alloantibodies with granulocytes were assessed by flow cytometry in 74 healthy blood donors and 6 persons with known variant FcgammaRIIIB genes. The granulocytes were tested with 1 NA1-specific MoAb, 1 NA2-specific MoAb, 4 NA1-specific alloantibodies, and 4 NA2-specific alloantibodies. RESULTS: Analysis of granulocytes from persons with variant NA genotypes found that single-base substitutions in FcgammaRIIIB at 141 and at 349 are important in NA1 expression and those at 227 and 277 are important in NA2 expression. Among blood donors, neither age, sex, nor race affected the expression of NA1 or NA2. The NA2-specific MoAb reacted more intensely with granulocytes from NA2-double-dose cells than with those from NA-single-dose cells, but this was not true for the NA2-specific alloantibodies. There was no difference in the reactions of the NA1-specific MoAbs and alloantibodies with donor samples of known NA1-double-dose or NA-single-dose cells. The intensity of reactions of both the NA1- and NA2-specific MoAbs and alloantibodies were strongly correlated on double-dose cells but not on single-dose cells. In fact, granulocytes from 7 healthy blood donors, phenotyped as NA-single-dose with the MoAbs, were phenotyped as NA2-double-dose with the alloantibodies. Variations in FcgammaRIIIB are common in blacks, but 5 of the 6 donors were white. These results suggest that FcgammaRIIIB variations may be common in both whites and blacks. CONCLUSIONS: NA2 expression is affected by polymorphisms in FcgammaRIIIB 227 and FcgammaRIIIB 277, both of which are involved in an FcgammaRIIIb N-glycosylation site. Polymorphisms in FcgammaRIIIB at 141 and 349 appear more important to NA1 expression.

Massive immune haemolysis after allogeneic peripheral blood stem cell transplantation with minor ABO incompatibility.

Immune haemolysis as a result of minor ABO incompatibility is an underappreciated complication of haematopoietic transplantation. The increased lymphoid content of peripheral blood stem cell (PBSC) transplants may increase the incidence and severity of this event. We observed massive immune haemolysis in 3 out of 10 consecutive patients undergoing HLA-identical, related-donor PBSC transplants with minor ABO incompatibility. Non-ablative conditioning had been given in 9 of these 10 cases, including two with haemolysis. Cyclosporin alone was used as prophylaxis against graft-vs.-host disease (GVHD). Catastrophic haemolysis of 78% of the circulating red cell mass led to anoxic death in the first case seen, but severe consequences were avoided by early, vigorous donor-compatible red cell transfusions in the subsequent two cases. Haemolysis began 7-11 d after PBSC infusion and all patients with haemolysis had a positive direct antiglobulin test (DAT), with eluate reactivity against the relevant recipient antigen. However, neither the intensity of the DAT, the donor isohaemagglutinin titre, nor other factors could reliably be used to predict the occurrence of haemolysis. Our data indicate that haemolysis may be frequent and severe after transplantation of minor ABO-incompatible PBSCs when utilizing cyclosporin alone to prevent GVHD. Meticulous clinical monitoring and early, vigorous donor-compatible red cell transfusions should be practiced in all instances.

RBC autoantibodies in autoimmune lymphoproliferative syndrome.

BACKGROUND: Patients with autoimmune lymphoproliferative syndrome (ALPS) have an autosomal dominant genetic defect that affects lymphocyte apoptosis and is associated with chronic nonmalignant lymphadenopathy, splenomegaly, and autoimmunity, particularly affecting RBCs, WBCs, and platelets. STUDY DESIGN AND METHODS: DATs were performed on 34 consecutive patients with ALPS and 37 of their clinically unaffected relatives. The effects of age, sex, race, and immunoglobulin levels on the incidence of autoantibodies and clinical hemolysis were assessed. RESULTS: The DAT was positive in 21 (62%) of ALPS patients but in only 1 (3%) of their relatives (p = 0.001). The DAT reacted because of IgG alone in 43 percent, complement alone in 5 percent, and IgG plus complement in 19 percent; 33 percent of the patients' cells had a positive reaction with polyspecific reagent only. All 10 ALPS patients with a history of hemolytic anemia had a positive DAT. Sixty percent of them had only IgG on their cells, 30 percent had IgG and complement, and 10 percent reacted only with polyspecific reagent. Of the 11 patients with a positive DAT and no history of hemolytic anemia, IgG alone was present in 27 percent, complement alone in 9%, and IgG plus complement in 9 percent; 55 percent had positive DATs only with polyspecific reagent. Among ALPS patients, those with a positive DAT had greater quantities of cells with increased alpha and ss T-cell receptors that phenotyped as CD4-CD8- and higher IgG levels. CONCLUSIONS: The DAT results in ALPS patients are most similar to those found in warm autoimmune hemolytic anemia. The DAT is useful to distinguish affected and unaffected persons within an ALPS family.

Autoantibody formation in the alloimmunized red blood cell recipient: clinical and laboratory implications.

Alloimmunization to erythrocyte antigens is a well-characterized complication in heavily transfused patients. Less well recognized, however, is the frequency of autoantibody formation in these previously alloimmunized patients. The autoantibodies are heterogeneous and of variable clinical significance. We describe the clinical history, laboratory evaluation, diagnosis, and treatment in 4 patients who developed autoantibodies in temporal association with alloantibody formation. In one case, the autoantibody found on routine screening had no clinical significance. In another case, the autoantibody made accurate blood typing and subsequent transfusion exceedingly difficult. Two patients experienced hemolysis as a consequence of the autoantibody. The management of both patients included supportive measures, while one patient required glucocorticosteroids and red blood cell transfusion. We review the published literature concerning autoimmunization in the transfused alloimmunized host. The spectrum of clinical consequences is important for the general practitioner to recognize, as these complications may occur during routine blood transfusions.

Detection of granulocyte antibodies by flow cytometry without the use of pure granulocyte isolates.

Established methods used to detect serum antibodies to granulocytes require the isolation of granulocytes. Flow cytometric analysis of granulocytes with monoclonal antibodies eliminates the need for granulocyte isolation. The purpose of this study was to develop a method to evaluate reactions of antibodies to granulocytes without separating granulocytes from other leukocytes. Three screening cell samples for granulocyte antibody detection were prepared from whole-blood samples in which the red blood cells (RBCs) were lysed and remaining leukocytes tested against sera at 4 degrees C. Binding of human alloantibodies to the screening cells was determined by flow cytometric analysis using phycoerythrin-conjugated antibody to human immunoglobulin. Forward and side scatter were used to analyze granulocytes separately from other leukocytes. The assay was validated by testing granulocytes with reference alloantibodies directed to NA1, NA2, 5b, and Mart antigens. Samples from 32 patients were tested, and the results of the assays were compared with the results of testing the samples in a granulocyte immunofluorescence (GIF) assay performed by a reference laboratory. In the whole-blood flow cytometric (WBFC) assay the mean fluorescence intensities of reference antisera with antigen-positive cells, expressed in arbitrary units, were anti-NA1 = 48 to 221, anti-NA2 = 24 to 69, anti-5b = 13 to 57, and anti-Mart = 42 to 72. In contrast, the mean fluorescence intensity of type AB-negative control sera ranged from 3 to 11. Of the 32 patient sera tested, 23 were positive (range = 12 to 56) and 9 were negative (range = 3 to 10). When compared with the results obtained by the reference laboratory, 27 sera were concordant between the WBFC and the GIF assays. Four of the samples were positive in WBFC (range = 11 to 31) and negative in GIF and one sample was negative in WBFC (range = 5 to 6) and positive in GIF. Leukocytes prepared from whole blood after lysis of RBCs can be used in flow cytometric analysis to detect granulocyte alloantibodies. The results of testing for granulocyte antibodies with this assay were similar to results of testing sera in GIF. Further comparative studies are indicated to confirm findings and explain the discordant results.

Intravenous Rh immune globulin prevents alloimmunization in D- granulocyte recipients but obscures the detection of an allo-anti-K.

Rh immune globulin (RhIG) has been used to prevent alloimmunization in D(-) recipients of apheresis platelet transfusions from D(+) donors that may contain up to 5 mL of D(+) red blood cells (RBCs). Granulocyte concentrates contain approximately 30 mL of RBCs and it has been necessary to give D(-) recipients granulocyte transfusions from D(+) donors. Intravenous RhIG has not yet been demonstrated to be effective in preventing D alloimmunization with granulocyte transfusions. Four D(-) recipients received multiple D(+) granulocyte transfusions from D(+) donors and multiple injections of intravenous RhIG at a standard dose of 600 microg for each D(+) transfusion. Two D(-) males with chronic granulomatous disease were given 32 and 13 daily granulocyte transfusions, 18 and 2 of which, respectively, were D(+). After the first dose of intravenous RhIG, both patients exhibited circulating anti-D that was undetectable 3 to 4 years later. Two patients with severe aplastic anemia were given 5 and 14 granulocyte transfusions, 4 and 7 of which, respectively, were D(+). Both patients died before the effectiveness of RhIG could be assessed. In one of these patients the indirect and direct antiglobulin tests became positive after the first dose of intravenous RhIG, which required that subsequent granulocyte transfusions from D(+) donors be crossmatched by immediate spin (IS) testing only. A delayed hemolytic reaction attributed to allo-anti-K occurred after granulocytes from a K(+) donor were given to this patient. These results suggest that intravenous RhIG can be used to prevent alloimmunization to D in D(-) patients receiving large quantities of RBCs from D(+) granulocyte transfusions. However, anti-D and other passive antibodies from RhIG prohibit the use of the antiglobulin crossmatch with antigen-positive granulocyte donor samples. It may be important to frequently collect new samples to screen for newly formed allo-antibodies when IS crossmatches are used in place of the antiglobulin crossmatch.

Description of serologic features in autoimmune lymphoproliferative syndrome.

BACKGROUND: Autoimmune lymphoproliferative syndrome (ALPS) is a recently recognized and rare disorder associated with inherited defects in the FAS: gene or other regulators of lymphocyte apoptosis. It is characterized by massive lymphadenopathy; splenomegaly; autoimmunity including episodes of immune hemolytic anemia, thrombocytopenia, and neutropenia.(1) The serologic basis for immune cytopenias associated with ALPS has not been previously characterized. STUDY DESIGN AND METHODS: RBC, granulocyte, and platelet serologies for ALPS patients and hepatitis C patients were assessed. Medical records were reviewed for clinical, immunologic, serologic, and transfusion history. Testing included: DAT; serum screening for antibodies to RBCs, granulocytes, platelets, cardiolipin, penicillin-coated RBCs, and human leukocyte antigens; antibody identification and IgG subclass; RBC phenotype. RESULTS: In a cohort of 11 patients with apoptosis defects (eight with heterozygous FAS: gene mutations); many had histories of hemolytic anemia (7), thrombocytopenia (4), and/or leukopenia (11); nine received steroid therapy, seven underwent splenectomy; five had been remotely transfused. On the basis of serologic testing even when they were clinically stable, nine had positive DATs; two had alloantibodies; 6 had IgG and/or IgM antibodies to cardiolipin; seven had platelet-directed antibodies; three had granulocyte-directed antibodies; none had HLA antibodies. CONCLUSIONS: Nearly all ALPS patients have antibodies directed against one or more hematopoietic cell lineages. Serologic testing is critical in the evaluation of these individuals and when transfusion is indicated, red cells that are matched for clinically significant C, E, and K antigens should be considered.

Variations in genes encoding neutrophil antigens NA1 and NA2.

BACKGROUND: Neutrophil-specific antigens NA1 and NA2 are located on Fcgamma receptor IIIb (FcgammaRIIIb). NA1 and NA2 forms of FcgammaRIIIb differ by four amino acids and the corresponding genes by five nucleotides. Variations in NA gene frequencies are encountered among ethnic groups. Altered forms of the genes are expected among individuals. STUDY DESIGN AND METHODS: RFLPs associated with four recognition sites were used to determine NA genotypes of 232 individuals. When atypical NA genotypes were identified, FcgammaRIIIB and FcgammaRIIIA regions were sequenced. RESULTS: NA1 FcgammaRIIIB frequency in 100 Japanese (0.66) was greater than that in 53 African Americans (blacks) (0.40; p<0.01) and 79 whites (0.32; p<0.001). Sequencing of atypical FcgammaRIIIB in 16 people confirmed that four blacks had G-->A substitutions at 227; 7 blacks had A-->G substitutions at 277; and 1 Japanese person had C-->G at 141 and G-->T at 227. A at 227 and G at 277 represent expected nts of NA1 FcgammaRIIIB. One black had an NA1 FcgammaRIIIB with a G-->A substitution at 349; A is normally found in NA2 FcgammaRIIIB at 349. Sequencing atypical FcgammaRIIIA in three persons revealed that two blacks had G-->A substitutions at 277 plus C-->A substitutions at 266 and 1 white had previously described T-->G at 230. Two blacks with atypical NA2 FcgammaRIIIB had T-->G FcgammaRIIIA at 230. One black was NA(null). CONCLUSION: NA2 FcgammaRIIIB is more polymorphic in blacks than in whites or Japanese persons. Chimeric FcgammaRIIIB alleles are most similar to NA2 FcgammaRIIIB. One alternate allele of NA1 (NA1*02) and four alternate alleles of NA2 (NA2*02, NA2*03, NA2*04, and NA2*05) are described.

Variations in the expression of granulocyte antigen NB1.

BACKGROUND: Between 87 and 97 percent of whites express NB1 alloantigen on some but not all of their granulocytes. The expression of NB1 has not been compared among large groups of adults of different sexes, ages, and ethnic groups. Previous testing of whites suggests that the expression of NB1 is variable. STUDY DESIGN AND METHODS: Serologic testing of granulocytes from 224 persons with two examples of MoAb to NB1 (1B5 and 7D8) was performed to distinguish phenotypic differences among age, sex, and ethnic groups and differences in reactivity to MoAbs. The donors were from 17 to 82 years of age, and 87 were female. They were from four ethnic backgrounds: 54 were African American (black), 10 were Asian, 9 Hispanic, and 152 white. Granulocytes were tested by flow cytometry. Parallel testing with MoAbs to CD16, CD11b, and CD45 served as controls. The size of the granulocyte population reacting with 1B5 and 7D8 and the respective mean, median, and peak cell fluorescence intensities were analyzed. RESULTS: The expression of 7D8 and 1B5 was greater on granulocytes from female donors. The expression of 7D8 fell in older women but not in men. There were no differences among the four racial groups in either the frequency of NB1 as determined by 1B5 or 7D8 or in the size of the population of granulocytes reacting with either antibody. When the fluorescence intensities of the antibody reactions were compared among groups, there were no differences in reactivity with 1B5. However, reactions with 7D8 were all greater in blacks. Comparison of the size of the antigen-positive granulocyte population, as determined by antibody reactivity, showed that only 30 donors differed by more than 10 percent. These discordant results were more likely to occur in whites than in blacks (18% vs. 4%, p<0.02). CONCLUSIONS: NB1 is composed of at least two epitopes as determined by serologic studies. The expression of both antigens is greater in females. The 7D8-reactive epitope appears to be more prevalent or more accessible on granulocytes of blacks. Variations in the expression of NB1 are more likely to occur in whites. The biochemical and molecular basis of these variations are not known.

Drug-induced hemolysis: cefotetan-dependent hemolytic anemia mimicking an acute intravascular immune transfusion reaction.

Numerous cases of drug-induced hemolytic anemia have been described in patients treated with penicillin or cephalosporin. Second and third generation cephalosporins are more commonly implicated in hemolytic reactions than first generation cephalosporins. We report a case of severe cefotetan-induced hemolytic anemia in a previously healthy 46-year-old woman undergoing an elective hysterectomy. The patient received 2 g of intravenous cefotetan intraoperatively and subsequently at 12 and 24 h post-operatively. She complained of diarrhea and fever on the third post-operative day and was seen in her gynecologist's office on the fifth post-operative day (hemoglobin = 10.5 g/dL). On the seventh post-operative day, she complained of fever and soreness around the suprapubic catheter site and was given a prescription for 500 mg oral cephalexin four times a day. The next day she was seen in the gynecologist's office and reported feeling better. Ten days after the operation her fatigue worsened and her hemoglobin was 4.8 g/dL. She was transfused with 3 units of packed red blood cells (PRBC) and was given 1 g of cefotetan intravenously. During the transfusion of the second unit of PRBC nursing staff observed gross hemoglobinuria and she subsequently developed acute renal failure. Laboratory chemistry parameters were consistent with severe acute hemolysis. The patient's direct antiglobulin test was reactive and her serum reacted with cefotetan-coated red blood cells (RBCs) and serum plus soluble cefotetan reacted with untreated RBCs. The titration endpoint of the serum against cefotetan-coated RBCs was 40,960, while the serum plus soluble cefotetan against uncoated RBCs was 2,560. This case of severe cefotetan-induced hemolysis was complicated by an acute hemolytic event that occurred during the transfusion of PRBC. Clinical and transfusion service staff must consider drug-induced hemolysis in the differential diagnosis of acute anemia.

Identification of the null HLA-A2 allele, A*0232N.

We have identified a null HLA-A*02 allele, HLA-A*0232N, by using a combination of serology, flow cytometry, polymerase chain reaction using sequence-specific primers (PCR-SSP) and full-length sequencing. The null HLA-A2 allele was identified in an Asian individual originally typed by serology as an apparently homozygous HLA-A3, B51. Subsequent genotyping by PCR-SSP identified the genotype as HLA-A*0201, *0301, B*51, Cw*1402. The serological type and lack of detectable HLA-A2 was confirmed using monoclonal antibody typing reagents. Flow cytometry studies failed to identify any cell surface HLA-A2 expression on the patient's peripheral blood lymphocytes. Genotyping using a PCR-SSP set designed to detect null alleles revealed the mutation had not been previously described. Full-length sequencing of the allele identified an allele which was subsequently named HLA-A*0232N. This allele is identical to HLA-A*0201 except for a novel point mutation (T for C) at position 493 which creates a premature stop codon. The sequencing enabled the development of a monospecific A*0232N PCR-SSP reaction which was used to screen 973 DNA samples: no further examples of A*0232N were identified.

Comparison of human platelet antigen (HPA)-1a typing by solid phase red cell adherence to HPA-1 allotypes determined by allele-specific restriction enzyme analysis.

Phenotype results for human platelet antigen (HPA)-1 by Capture-P(R), (Immucor, Inc., Norcross, GA) solid phase red cell adherence (SPRCA) were compared to results of allele-specific restriction enzyme analysis (ASRA) for the determination of HPA-1 allotype. Because the expression of HPA-1a and HPA-1b is determined by a single nucleotide substitution of thymine --> cytosine at position 196 of the gene encoding membrane glycoprotein (GP)-IIIa, it is possible to distinguish the alternate forms of the gene using ASRA. Primers (5'- GCTCCAATGTACGGGGTAAACTC-3' and 5'-CAGACCTCCACCTTGTGCTCTATG- 3') were designed to amplify the region of DNA that contains the polymorphism and a restriction enzyme (Nci I) was used to cleave the DNA in a predictable manner. Platelet-rich plasma for immunophenotying and anticoagulated whole blood for DNA extraction were obtained from 159 platepheresis donors. Of 159 SPRCA tests, 138 were valid and 21 were invalid due to positive autologous controls. For 135 HPA-1a-positive and 2 HPA-1a-negative phenotype tests the DNA typing results correlated: 135 positive samples were either HPA-1a/a or HPA-1a/b and 2 negative samples were HPA-1b/b. One donor that typed as HPA-1b/b by ASRA had a positive result of 2+ on SPRCA. This donor had been previously typed by SPRCA as HPA-1a-negative and DNA typed as HPA-1b/b by our laboratory. Based on these findings results of = 3+ by SPRCA are interpreted as HPA-1a-positive for donor screening purposes. SPRCA test results of = 2+ are considered equivocal and the HPA-1 allotype is determined by ASRA. HPA-1a-negative donors by SPRCA must be confirmed as HPA-1b/b by ASRA prior to issue for a patient that requires HPA-1anegative platelets.

Granulocyte storage and antigen stability.

BACKGROUND: Current methods for the detection of granulocyte antibodies require panels of freshly isolated cells. This makes these assays time-consuming, costly, and technically difficult. STUDY DESIGN AND METHODS: The immunofluorescence method of detecting the binding of antibodies to granulocytes was modified for use with a flow cytometer, and methods were tested to store granulocytes for use in that assay. Granulocytes were stored at 4 degrees C for 7 days under three conditions: 1 -percent formaldehyde-fixed cells were stored in Hanks' balanced salt solution (HBSS); untreated cells were stored in tissue culture medium (RPMI-1640); and cells were fixed and stored with a commercial white cell-storage solution (Cyto-Chex Reagent). Antigen stability was evaluated by using monoclonal antibodies (MoAbs) and alloantibodies. Serologic studies were done by an indirect immunofluorescence assay and assessed by flow cytometric analysis. RESULTS: On Day 2, only 2 to 7 percent of granulocytes stored in RPMI-1640 remained. On Day 7, 67 to 76 percent of granulocytes fixed in formaldehyde and stored in HBSS remained, and 47 to 87 percent of granulocytes stored in a white cell-storage solution remained. All antigens were detectable by the MoAbs and alloantisera on Day 7. However, nonspecific staining by the fluorescein isothiocyanate (FITC)-conjugated secondary antibody hindered interpretation of test results on Day 4. Non-specific staining occurred over time and was associated with increased cell permeability during storage. Two sources of nonspecific staining were identified. The first source was the FITC-conjugated secondary antibody; it was eliminated by switching to a phycoerythrin conjugate. The second source was factors in human serum; it was resolved by examining only viable, impermeable cells identified by using 7-aminoactinomycin-D. CONCLUSION: Granulocytes and their antigens can be preserved for at least 7 days, but evaluation of antibody reactions was possible for only 4 days as a result of non-specific staining due to enhanced membrane permeability of dying cells.