The intra- and inter-assay variation of the indirect mixed antiglobulin reaction test: is a quality control suitable?
The test most commonly used to detect sperm antibodies is the mixed antiglobulin reaction (MAR), standardized by the World Health Organization. The indirect MAR test detects soluble sperm antibodies in seminal plasma by using healthy donor spermatozoa as antigen. In this study we systematically investigated the influence of donor spermatozoa and the source of sperm antibodies upon the results of the indirect MAR test, and calculated the intra- and inter-assay variations. Using one individual seminal plasma and the same donor semen, results of the indirect MAR test are highly reproducible (low intra-assay variation). Two dimensions of inter-assay variation must be considered: (i) serial ejaculates of an individual donor may be used at different times; (ii) different donors may be applied to identical antibody sources. Donor spermatozoa strongly influenced the results of the indirect MAR test. Using multivariate statistical tests, highly significant main effects between the different donors (P < 0.001) and specific reciprocal effects between donor spermatozoa and seminal plasma samples (P < 0.001) were observed. The high inter-assay variation of the indirect MAR test will lead to incorrect results. There is urgent need of a reliable and reproducible test for sperm antibody detection to improve quality control of the methods. (+info)
A persistent severe autoimmune hemolytic anemia despite apparent direct antiglobulin test negativization.
BACKGROUND AND OBJECTIVE: Not all cases of autoimmune hemolytic anemia (AIHA) are diagnosed by the direct antiglobulin test (DAT). We present and discuss a simple method of enhancing the sensitivity of the standard DAT. DESIGN AND METHODS: We report the case of a five-month-old child diagnosed with a severe IgG-mediated AIHA, characterized by quick DAT negativization despite clinical worsening. Warm AIHA with negative DAT, possibly due to a low affinity autoantibody, unresponsive to conventional therapy, was hypothesized. RESULTS: The DAT resulted strongly positive with anti-IgG serum using a 4C saline for erythrocyte washing, to reduce the dissociation of the supposed low affinity autoantibody. Very intensive cytoreductive treatment was administered twice until clinical remission was obtained. INTERPRETATION AND CONCLUSIONS: The clinical course of AIHA can be dissociated by the DAT. Since autoantibody-mediated hemolysis with negative DAT rarely occurs, once other causes of high reticulocyte count anemia have been ruled out, the DAT after ice-cold saline washing could be a useful and easy means of corroborating the diagnosis of AHIA, when traditional methods fail. (+info)
Posttreatment follow-Up of brucellosis by PCR assay.
In order to evaluate the usefulness of a peripheral blood PCR assay in the posttreatment follow-up of brucellosis, a cohort of 30 patients was studied by means of blood cultures, rose Bengal, seroagglutination, Coombs' antibrucella tests, and PCR assay at the time of diagnosis, at the end of treatment, and 2, 4, and 6 months later. Of the 29 patients whose PCR assays were initially positive, 28 (96.5%) were negative at the conclusion of the treatment. PCR was positive for the two patients who had relapses and negative for another four who had suspected but unconfirmed relapses. PCR was negative for 98.3% of the follow-up samples from those patients who had a favorable evolution. In conclusion, PCR appears to be a very useful technique, not only for the initial diagnosis of the disease, but also for posttreatment follow-up and the early detection of relapses. (+info)
Relative sensitivity of direct antiglobulin test, antibody's elution and flow cytometry in the serologic diagnosis of immune hemolytic transfusion reactions.
BACKGROUND AND OBJECTIVE: Current immunohematology practice dictates that serologic diagnosis of immune hemolytic transfusion reactions (IHTR) is based on the finding of a positive post-transfusion direct antiglobulin test (DAT). However, since DAT may fail to detect antibody-coated cells when they constitute a minor population amid a large number of non-sensitized ones, we investigated whether antibody detection in eluates or by flow cytometry is more sensitive than DAT in this context. DESIGN AND METHODS: Ten samples of red blood cells sensitized with allo- or autoantibodies were diluted in non-sensitized red blood cells to final concentrations ranging from 10% to 0.1%. DAT, antibody detection in eluates, and immunofluorescence by flow cytometry were performed on each mixture. RESULTS: DAT failed to detect sensitized cells in all but two cases in that only the 10% dilution yielded a positive DAT. Antibody detection in eluates and by flow cytometry was able to detect up to 1% sensitized cells in most cases. INTERPRETATION AND CONCLUSIONS: Antibody detection in eluates and by flow cytometry is more sensitive than DAT for detecting minor populations of IgG-coated cells. These techniques should be included in the routine investigation of suspected cases of IHTR. (+info)
Allogeneic peripheral blood hematopoietic stem cell transplantation: guidelines for red blood cell immuno-hematological assessment and transfusion practice.Societe Francaise de Greffe de Moelle.
Allogeneic peripheral blood hematopoietic stem cell transplantation (PBSCT) is presently being evaluated in a French randomized study comparing peripheral blood vs bone marrow. Cases of potentially lethal acute hemolysis have recently been reported after allogeneic PBSCT in the presence of a 'minor' ABO incompatibility. Patients were frequently transfused with recipient-compatible and donor-incompatible RBC and usually did not receive methotrexate in addition to cyclosporin A for graft-versus-host disease (GVHD) prophylaxis. In order to homogenize immuno-hematological (IH) assessment and transfusion practices within our protocol, we made proposals to 25 allo-transplant French centers on the following aspects: pre-inclusion IH assessment, IH exclusion criteria, transfusion rules, post-transplant IH surveillance and treatment of hemolysis. Analysis of responses to our proposals led to the elaboration of guidelines which were approved and implemented by the French Bone Marrow Transplantation Society (SFGM). Pre-inclusion IH testing includes mandatory detection and titration of anti-RBC allo-Ab, as well as titration of anti-A and anti-B Ab. The presence in the donor of an anti-A (group A or AB recipients), anti-B (group B or AB recipients) Ab with a titer >1/32 or the presence of allo-Ab against Rh, Kell, Fya, Fyb, Jka, Jkb, Ss Ag present on recipient RBC is an exclusion criterion for the protocol. ABO and RhD compatibility of RBC blood products with both HSC donor and recipient is mandatory. A similar compatibility is also required for Rh (other than D) and Kell Ag. If not possible, compatibility of RBC blood products with the HSC donor is mandatory. Lastly, guidelines regarding post-transplantation IH follow-up as well as acute hemolysis treatment have been elaborated. The implementation of these guidelines should contribute to enhancing the quality of transfusion practice after PBSCT. Such an approach will be applied to other aspects of transfusion medicine in the setting of HSC transplantation. Bone Marrow Transplantation(2000) 25, 507-512. (+info)
Unrelated umbilical cord blood transplantation in infancy for mucopolysaccharidosis type IIB (Hunter syndrome) complicated by autoimmune hemolytic anemia.
This report describes unrelated umbilical cord blood transplantation for a 10-month-old infant boy with mucopolysaccharidosis IIB (Hunter syndrome), an X-linked metabolic storage disorder due to deficiency of iduronate sulfatase. Two years after transplant approximately 55% normal plasma enzyme activity has been restored and abnormal urinary excretion of glycosaminoglycans has nearly completely resolved. The boy has exhibited normal growth and development after transplant. Nine months after transplant he developed severe autoimmune hemolytic anemia and required 14 months of corticosteroid treatment to prevent clinically significant anemia. Bone marrow transplantation for Hunter syndrome and post-transplant hemolytic anemia are reviewed. Bone Marrow Transplantation (2000). (+info)
Modification of the immunogenicity and antigenicity of rat hepatoma cells. I. Cell-surface stabilization with glutaraldehyde.
gamma-Irradiated rat hepatoma cells are immunogenic in syngeneic WAB/Not rats, so that immunized animals are protected against tumour-cell challenge and circulating tumour-specific antibody is produced. Treatment of the immunizing cells with glutaraldehyde at concentrations of 0.001% or greater for 30 min rendered these cells non-protective in tumour-rejection tests and no longer able to induce significant formation of specific antibody. However, tumour-specific antigens were shown to be expressed upon treated cells; they specifically bound tumour-specific antibody from syngeneic immune sera assessed in indirect membrane-immunofluorescence tests. Also, these cells specifically absorbed antibody from immune or tumour-bearer sera, as demonstrated in the indirect membrane-immunofluorescence test or a complement-dependent 51Cr-release test. Alloantigen expression was not influenced by glutaraldehyde treatment, although glutaraldehyde-treated hepatoma cells failed to induce alloantibody formation in KX/Not rats. Polyacrylamide-gel electrophoresis of treated cells, surface-labelled with 125I, indicated that extensive cross-linking of the surface protein occurred as a result of glutaraldehyde treatment. The present findings establish that although the expression of a tumour-specific antigen is necessary for the induction of immuno-protection against tumour-cell challenge, this alone is not a sufficient condition for eliciting tumour immunity. (+info)
Poly IC therapy in aleutian disease of mink.
Twenty-four virgin female aleutian mink were infected with aleutian disease agent and after 24 hours, 12 of these were treated with a course of polyinosinic acid-polycytidilic acid (Poly IC) injections. After six weeks the gammaglobulin level was significantly lower in the treated group but at 12 weeks this difference was no longer present. Four of the treated mink had normal target organ histology when killed at 20 weeks. The untreated group all showed moderate to marked changes but this difference was not statistically significant. There was a marked increase in the reactive lymphocyte blastogenesis index during the first weeks of infection and the phytohaemagglutinin response was seen to fall progressively. The antiglobulin reaction usually became positive after infection but neither antinuclear nor antierythrocyte antibodies were found. Precipitating antibodies to several polynucleotides were frequently present and were unrelated to infection or to Poly IC treatment. (+info)