Luminex® Single Antigens

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  • In your laboratory, if the result of single antigen(One lambda) is more than 20,000 MFI, serum must 1:8 dilute with RPMI, right?

    Can you tell me why did you come to that decision?

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  • Using real data for a virtual crossmatch

    Andrea A. Zachary*, Jeffrey T. Sholander, Julie A. Houp, Mary S. Leffell
    Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

    Article history:
    Received 8 April 2009
    Accepted 9 June 2009
    Available online 12 June 2009

    Abstract

    Virtual crossmatches have been performed for more than 40 years under the guise of unacceptable antigens. Today, solid-phase assays provide the opportunity for more accurate identification and more precise mea- surement of the strength of donor-specific antibodies. The process of performing a virtual crossmatch begins with establishing a correlation between the antibody testing assay and the results of actual crossmatches. We provide here data indicating that the identity and strength of DSA defined with solid-phase phenotype panels correlates significantly with the outcome of both cytotoxic (CDC; r 0.83) and flow cytometric (r 0.85) crossmatches. Based on the threshold established from these correlations, we were able to correctly predict the results of CDC and flow cytometric crossmatches in 92.8 and 92.4% of cases, respectively. The correlations with single antigen panels were substantially lower (82.6 – 47.9%) and may be caused by a variety of factors, including variability in the amount and condition of different antigens and extremely high sensitivity, which may make the test less robust. We demonstrate that adding additional information to the solid-phase results can increase the frequency of correct crossmatch prediction. We also present data demonstrating an addi- tional use of the virtual crossmatch in posttransplant monitoring.
    © 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

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  • Mary S. Leffell and Andrea A. Zachary

    Department of Medicine, Johns Hopkins University, School of Medicine, Baltimore, Maryland, USA
    Correspondence to Mary S. Leffell, PhD, Immunogenetics Laboratory, Department of Medicine, Johns Hopkins University School of Medicine, 2041 E. Monument Street, Baltimore, MD 21205, USA Tel: +1 410 955 3600; fax: +1 410 944 0431; e-mail: msl@jhmi.edu
    Current Opinion in Organ Transplantation 2010, 15:2 – 7

    Purpose of review
    Solid-phase immunoassays increase the accuracy of assessing pretransplant immunologic risk and facilitate posttransplant prediction and diagnosis of antibody- mediated rejection (AMR). This review will describe methods available for antibody analyses, discuss the types of targets of AMR and the characteristics of pathogenic alloantibodies, and provide guidelines for the application of antibody tests in the prediction of rejection risk and diagnosis of rejection.
    Recent findings
    The presence of human leukocyte antigen-specific antibodies increases the risk of AMR, but the clinical relevance of low antibody levels is questioned with reports of stable graft function in their presence. Posttransplant monitoring has been shown to provide early diagnosis of AMR permitting preemptive intervention. Antibodies to other alloantigens and autoantigens are being implicated as potential targets for both acute and chronic AMR. Certain limitations and interfering factors have also been recognized that should be recognized in the interpretation of solid-phase antibody assay results.
    Summary
    Contemporary technology is clearly advancing the detection of various antibodies that can contribute to AMR, but continued work is needed to elucidate the relevance of very low levels of human leukocyte antigen-specific antibody and the importance of antibodies to other alloantigens and autoantigens.
    Keywords alloantibodies, antibody-mediated rejection, autoantibodies, donor-specific antibodies, human leukocyte antigen-specific antibodies
    Curr Opin Organ Transplant 15:2 – 7
    © 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins 1087-2418

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  • Paul Sikorski a,*, Dessislava Kopchaliiska b, Donna P. Lucas a, Mary S. Leffell a, Andrea A. Zachary a

    a The Johns Hopkins Immunogenetics Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
    b Immunogenetics Laboratory. Department of Surgery, University of California at San Francisco, San Francisco, California, USA

    Article history:
    Received 13 May 2010
    Accepted 6 October 2010
    Available online 21 October 2010
    Keywords: HLA-A36 A36

    Abstract
    Humoral sensitization to HLA often results in antibodies to public determinants shared among two or more antigens. Although monoclonal antibodies to A36 have been produced, there are no reports of polyclonal antibodies that react with A36 but not A1. We report here sera from a heart transplant recipient that reacted with A36 but not A1 in tests with both phenotype and single antigen panels on the Luminex platform. Flow cytometric crossmatch tests yielded positive results with an A36 bearing phenotype but not with a pheno- type containing A1. A36 reactivity in solid phase assays was abrogated by absorption with cells bearing A36, but not with A1-positive cells. The frequency of B cells in this patient specific for A1 was comparable to that for individuals not sensitized to A1. These data indicate that reactivity was to an epitope present on A36 but absent from A1.

    © 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

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  • Andrea Zachary a and Nancy L. Reinsmoen b

    a John Hopkins University, Immunogenetics Laboratory, Baltimore, Maryland and b Cedars-Sinai Medical Center, HLA & Immunogenetics Laboratory, Los Angeles, California, USA
    Correspondence to Nancy L. Reinsmoen, PhD, Director, HLA & Immunogenetics Laboratory, Cedars- Sinai Medical Center, 8723 Alden Drive, SSB 197, Los Angeles, CA 90048, USA Tel: +1 310 423 4979; e-mail: nancy.reinsmoen@cshs.org Current Opinion in Organ Transplantation 2011, 16:410 – 415

    Purpose of review
    Two major desensitization protocols have been used to eliminate or reduce HLA antibodies to a level that allows transplantation with a low risk of antibody-mediated rejection (AMR). This review will focus on the antibody testing methods used to assess changes in the breadth and strength of antibody levels and the relative strength of donor HLA-specific antibodies (DHSAs).
    Recent findings
    Correlations of solid-phase immunoassay (SPI) class I and II levels with the donor- specific T and B cross-match results have shown the acceptable levels of DHSA that correlate with a low risk for AMR. The DSHA levels determined by SPI correlate with cross-match results and with clinical outcome. Therefore, the results of either assay can be used to determine the risk of AMR and when treatment has reduced DSHA to a level safe for transplantation. Monitoring DSHA is important for guiding the number of treatments as well as the timing of additional treatments needed to achieve these acceptable levels.
    Summary
    DSHA monitoring, in both protocols, uses the correlation of solid-phase antibody testing and the donor-specific cross-match to determine the efficacy of the protocol and when the acceptable level of DSHA is achieved permitting transplantation with minimal likelihood of AMR.
    Keywords crossmatch, desensitization, donor HLA-specific antibody, IVIg, solid-phase immunoassays
    Curr Opin Organ Transplant 16:410 – 415
    © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins 1087-2418 

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  • Carly  J. Callender a,*, Marcelo Fernandez-Vina b, Mary S. Leffell a, Andrea A. Zachary a

    a The Johns Hopkins Immunogenetics Laboratory, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
    b Stanford University School of Medicine, Department of Pathology/Blood Center, Palo Alto, CA 94304, USA

    Article history:
    Received 14 July 2011
    Accepted 7 November 2011
    Available online 23 November 2011

    Keywords: HLA-DP DP CREG

    Abstract

    Clinical studies have demonstrated that HLA-DP-specific antibodies can be detrimental to a transplanted kidney. The number of patients affected is proportional to the frequency of DP antibodies.Wedetermined the frequency of HLA-DP-specific antibodies en toto and in the absence of cross-reactive DR antibodies. Of 650 waitlisted renal patients, 271 (42%) were reactive with HLA-DP antigens in solid-phase immunoassays. Of these 271 sera, 58 (21%) were negative for reactivity with cross-reactive DR antigens, and 16 (5.9%) had no class II antibody other than DP. Eliminating sera containing DR cross-reactive antibodies reduced the frequency but not the overall strength of DP antibodies. Although most DP antibodies were not expected to yield a positive cytotoxicity crossmatch, 2 DP-specific antibodies yielded cytotoxic crossmatch tests with titers of 512. The occurrence of HLA-DP-specific antibody differed significantly between previously transplanted (62%) and nontransplanted (38%) patients, but no difference was observed among patients categorized by race or sex. One serum demonstrated strong cross-reactivity between DP and DRB1*01:03 in the absence of DR1 or DR11 reactivity. Sequence alignments were performed and a possible new cross-reactivity between DRB1*01:03 and DP2, DP9, DP10, DP13, DP16, and DP17 was defined. Two additional sera confirmed this cross-reactivity.

    2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

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