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Therapeutic Human IgG Preparations Contain Mixture of HLA Antibodies to Native HLA Antigens and Cryptic Epitopes With Little Clinical Significance

Mangiola, Massimo, PhD1; Marrari, Marilyn, BA2; Ensor, Christopher, PharmD3,4; Spycher, Martin O., PhD5; Berger, Mel, MD6; Zeevi, Adriana, PhD1

doi: 10.1097/TP.0000000000002312
Original Clinical Science—General

Background Human immunoglobulins (H-Ig) are widely used in solid organ transplantation for immunoglobulin G (IgG) replacement and for desensitization and treatment of antibody-mediated rejection. They are obtained from plasma pools and may contain HLA antibodies that can be detrimental to transplant recipients. The goal of this study was to evaluate HLA antibodies in multiple lots of 2 commercial H-Ig preparations by Luminex single-antigen bead (SAB) and cell-based crossmatch assays.

Methods Thirty lots of 2 commercial H-Ig products (CSL Behring, King of Prussia, PA) were evaluated: 6 Hizentra and 24 Privigen. All were adsorbed and diluted 1:10 before testing. HLA IgG antibodies were determined by 2 Luminex SAB kits and C1q screen for complement-binding capability. Lots were tested for the presence of antibody to denatured vs. intact class I HLA alleles using acid-treated SAB. Surrogate T and B-cell flow cytometry crossmatches (FCXM) were performed with peripheral blood lymphocytes from 2 healthy donors.

Results Twenty-two (73%) lots at 1:10 showed SAB reactivity with mean fluorescent intensity of 2000 or greater for HLA class I, 67% (20/30 lots) for class II. The reactivity pattern was similar using both SAB kits. Acid treatment revealed antibodies to denatured class I: the majority of HLA-C, half of HLA-B and few HLA-A alleles. No C1q reactivity was observed. Surrogate flow cytometry crossmatch results were positive (>150 median channel shift), but were fourfold to eightfold lower than expected.

Conclusions The H-Ig products tested consisted of low titer, non–complement-binding HLA class I and class II antibodies; most of the observed class I HLA reactivity was toward denatured HLA antigens.

Thirty-two lots from 2 different commercial preparations of IVIg contain non-complement binding DSA with a MFI greater that 2000 MFI in about 2/3 of cases, the majority of which was directed against denatured antigens.

1 Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA.

2 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA.

3 Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA.

4 Department of Pharmacy and Therapeutics, University of Pittsburgh School of Pharmacy, Pittsburgh, PA.

5 CSL Behring AG, Bern, Switzerland.

6 CSL Behring LLC, King of Prussia, PA.

Received 31 January 2018. Revision received 1 May 2018.

Accepted 23 May 2018.

M.M. received support from CSL Behring LLC in form of reagents and supplies to conduct research. M.M. received support from CSL Behring LLC in form of reagents and supplies to conduct research. C.E. received support from CSL Behring LLC in form of reagents and supplies to conduct research. M.O.S. was employed by CSL Behring AG. M.B. was employed by CSL Behring LLC. A.Z. received support from CSL Behring LLC in form of reagents and supplies to conduct research.

M.M., M.M., A.Z. participated in research design. M.M., M.M., C.E., M.O.S., M.B., A.Z. participated in writing the article. M.M., M.M., A.Z. participated in the performance of the research. M.O.S., M.B. contributed new reagents or analytic tools. M.M., M.M., A.Z. participated in the data analysis.

Correspondence: Massimo Mangiola, PhD, University of Pittsburgh Medical Center, Pittsburgh, PA. (mangiolam@upmc.edu).

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