Functional assessment of therapeutic anti‐D immunoglobulin using Fc‐mediated assays

Abstract

Therapeutic anti-D immunoglobulin preparations issued by the Scottish National Blood Transfusion Service, between 1980 and 1986, were evaluated using in-vitro Fc-mediated functional tests that reflect potential in-vivo mechanisms of specific red cell destruction and clearance. All batches tested were found to: (a) contain anti-D of mainly IgG1 subclass and lesser amounts of IgG3; (b) mediate lymphocyte and monocyte rosetting; and (c) produce lytic activity in both K cell and monocyte ADCC. The functional activity of the therapeutic immunoglobulin preparations over this period of production had not altered despite increased plasma contributions latterly to the pool from deliberately immunized male donors. This is the first in-vitro study of the Fc-mediated function of therapeutic polyclonal anti-D preparations. As these preparations were clinically effective in the prophylactic anti-D programme, such bioassays of FcRI/II and FcRIII activity are justified for the future evaluation of immune plasma before blending for fractionation and production of therapeutic anti-D immunoglobulin.

Use of the DAF Assay to Assess the Functional Properties of Polyclonal and Monoclonal Rh D Antibodies

“…Individual variations were obtained in the ADCC assay with PBMCs collected from various donors. Such observations have already been described [11][12][13]. This heterogeneity could be due to a variation ofthe percentage oflymphocytes and monocytes between different PBMC preparations.…”

“…To investigate these mechanisms, different techniques have been developed which measure the in vitro interactions between sensitized RBCs and peripheral blood mononuclear cells (PBMCs): (1) visual assessment of rosette formation; (2) evaluations of the RBC lysis by measuring the release of radioactive chromium incorporated into RBCs [8][9][10][11]; (3) measurements of phagocytosis by chemiluminescence [12][13][14]. More recently, different colorimetric assays were developed.…”

“…Vox Sang 1996;71: [30][31][32][33][34][35][36] Ducrot/Beliard/Glacet/KleiniHarbonnier/ Benmostefa/Bourel expression of these Fc receptors between different donors have already been described [13]. Accordingly, a pool of 3 donations was generally used as effector cells to limit the variations between the tests.…”

Use of the DAF Assay to Assess the Functional Properties of Polyclonal and Monoclonal Rh D Antibodies

“…Such observations have already been de scribed [11][12][13], This heterogeneity could be due to a var iation of the percentage of lymphocytes and monocytes be tween different PBMC preparations. In addition, the effec tor cells express different types of Fey receptors [21][22][23][24] which have various functions [25,26] and variations of the expression of these Fc receptors between different donors have already been described [13]. Accordingly, a pool of 3 donations was generally used as effector cells to limit the variations between the tests.…”

Flow–Cytometric Method for the Quantitation of the Fc Function of Intravenous Immunoglobulin Preparations

“…These assays of Fc function either measure rosette formation or lytic activity caused by the antibody–dependent cellular toxicity (ADCC) of lymphocytes or monocytes against Rh–D–positive cells. Most ADCC assays, require radioactive labelling of red blood cells [11]though technical variations of this assay detect haemolysis by the pseudoperoxidase activity of the free haemoglobin [12]. All such assays are subject to interlaboratory variability as assay conditions are difficult to standardise.…”