High background in Luminex® assay for HLA antibody screening: Interest of Adsorb Out™

Zerrouki, A.; Ouadghiri, S.; Benseffaj, N.; Rachid, R.; Essakalli, M.

doi:10.1016/j.trim.2016.03.001

Cited by 6 publications

(11 citation statements)

References 27 publications

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“…All the IVIg lots reacted with the negative control beads coated with albumin ( Figure 3), indicating that the Moroccan IVIg preparations contain naturally occurring anti-albumin antibodies, almost a similar inference can be derived from a previous report monitoring anti-HLA antibodies of Moroccan sera [44]. Among the four IVIg lots, lot #1 showed the highest anti-albumin reactivity, followed by lot # 2, then lot # 3, and finally lot # 4 showed the least reactivity.…”

Section: Anti-albumin Reactivity Of the Four Ivig Lotssupporting

confidence: 81%

Characterization of the Anti-HLA Class I and II IgG Antibodies in Moroccan IVIg Using Regular Beads and Ibeads in Luminex Multiplex Single Antigen Immunoassay

Hilali¹

2017

IJI

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Section: Anti-albumin Reactivity Of the Four Ivig Lotssupporting

confidence: 81%

Characterization of the Anti-HLA Class I and II IgG Antibodies in Moroccan IVIg Using Regular Beads and Ibeads in Luminex Multiplex Single Antigen Immunoassay

Hilali¹

2017

IJI

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“…Since SAB assays are using undiluted serum—in contrast to the majority of solid‐phase immuno‐assays—high quantities of albumin, irrelevant immunoglobulins, haptoglobin, transferrin, antitrypsin, and fibrinogen can interfere in two ways: (i) by masking the binding of specific HLA antibodies and (ii) by nonspecific binding especially to the negative control bead . The latter effect can be partially removed by Adsorb Out ™ beads .…”

Section: Technical Challenges and Limitationsmentioning

confidence: 99%

Caveats of HLA antibody detection by solid‐phase assays

2019

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All authors contributed equally to the review SUMMARY Solid-phase assays for human leukocyte antigens (HLA) antibody detection have clearly revolutionized the field of HLA diagnostics and transplantation. The key advantages are a high sensitivity and specificity for detection of HLA antibodies compared with cell-based assays, as well as the potential for standardization. Solid-phase assays enabled the broad introduction of tools such as "virtual crossmatching" and "calculated panel reactive antibodies," which are essential components in many organ allocation systems, kidney-paired donation programs, and center-specific immunological risk stratification procedures. The most advanced solid-phase assays are the socalled single antigen beads (SAB). They are available now for more than 15 years, and the transplant community embraced their significant advantages. However, SAB analysis and interpretation is complex and many pitfalls have to be considered. In this review, we will discuss problems, limitations, and challenges using SAB. Furthermore, we express our wishes for improvements of SAB as well as their future use for immunological assessment and research purposes.

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“…These results were confirmed by Goggins and associates, 18 who found that Adsorb Out bead pretreatment reduced NC values to a normal range in 57% of serum samples. In addition, our previous study 19 demonstrated that NC values were decreased in 62.2 % of serum samples. It is known that the Adsorb Out reagent reduces the non-specific binding to latex beads.…”

Section: Discussionmentioning

confidence: 82%

“…This effect could be due to the mechanism of action of these pretreatments, which were confirmed in our previous study for Adsorb Out. 19 In the literature, multiple patient-specific factors may contribute to high levels of nonspecific background, including disease type, current drug/immunosuppressive therapy, and presence of hyperlipidemia, but most are unknown. 21 To identify the main reason of the high NC beads when screening for HLA antibodies in patient sera, we compared patients versus controls according to several characteristics, including drugs taken at time of analysis and kidney disease.…”

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confidence: 99%

Untitled

Zerrouki

Ouadghiri²,

Benseffaj³

et al. 2018

Exp Clin Transplant

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Objectives: The Luminex technology is the most sensitive diagnostic method for HLA antibody detection and identification. However, the interpretation of immunoassays is commonly affected by the artifact, and non-specific background. Sera from some patients show high negative control bead (NC) value, which makes assessing and interpretation of HLA antibodies difficult. In this study, we evaluated the effect of Adsorb Out reagent, dithiothreitol (DTT), and Ethylenediaminetetraacetic acid (EDTA) on the NC median fluorescence intensity value by comparing treated versus untreated patient sera. In addition, we wanted to identify whether kidney disease and administered medication influenced high NC median fluorescence intensity values by comparing patient versus control results. Materials and Methods: HLA antibody screening was performed on 3500 serum samples. Sera were analyzed using the standard protocol for Luminex antibody screening. Sera with high NC values were preincubated with Adsorb Out, DTT, and EDTA. Screening of these sera was then performed. Results: We found that 4% of samples showed high NC values. Adsorb Out, DTT, and EDTA decreased the NC values at 723.5 (299.25-1443) versus 85 (34-218; P < .001), at 723.5 (299.25-1443) versus 184 (106-597; P < .001), and at 723.5 (299.25-1443) versus 455 (131-1177; P = .004). These succeeded in bringing back NC values to normal range in 69.2%, 43%, and 30% of treated sera, respectively. In addition, the differences of corticoids, immunosuppressive, and heparin drugs between patients and controls were statistically significant (P < .001, < .001, and = .043). However, presence of kidney disease was not significant between these groups. Conclusions: All pretreatments had an important effect in decreasing negative control values, with Adsorb Out having highest efficiency. Serum-specific components could contribute to high negative control bead median fluorescence intensity values. Further studies are needed to determine the adequate pretreatment of patient sera. Key words: Adsorb Out, Dithiothreitol, Ethylenediaminetetraacetic acid, HLA antibodies, Luminex IntroductionLuminex technology is a sensitive flow cytometric assay platform that relies on soluble or recombinant HLA molecules bound to polystyrene microspheres. These are currently the standard beads used for antibody screening and identification. 1-3 This technology utilizes a population of up to 100 unique beads that are distinguishable by a unique color address created by 2 embedded red fluorescent dyes, giving each bead a unique signal ratio. Each bead is coated with multiple copies of recombinant HLA antigen. The assay detects the relative level of immunoglobulin G (IgG) antibody bound to each bead using a second phycoerythrin fluorescence-labeled antihuman IgG antibody. The fluorescent intensity is represented as the mean fluorescence intensity (MFI) value, the amount of HLA-antigen-specific IgG antibody bound to each bead corresponding to a nonstandardized measure of relative fluorescence intensity. 4 ...

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High background in Luminex® assay for HLA antibody screening: Interest of Adsorb Out™

Cited by 6 publications

References 27 publications

Characterization of the Anti-HLA Class I and II IgG Antibodies in Moroccan IVIg Using Regular Beads and Ibeads in Luminex Multiplex Single Antigen Immunoassay

Characterization of the Anti-HLA Class I and II IgG Antibodies in Moroccan IVIg Using Regular Beads and Ibeads in Luminex Multiplex Single Antigen Immunoassay

Caveats of HLA antibody detection by solid‐phase assays

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