Anti-D Immunoglobulin Preparations: the Stability of Anti-D Concentrations and the Error of the Assay of Anti-D

Abstract. An analysis of the assay of 28 preparations of anti-D immunoglobulin using a radioisotope method carried out at 6-monthly intervals for 2–4.5 years showed an average fall in anti-D concentration ot 10.6% each year, with 99% confidence limits ot 6.8–14.7%. The fall in anti-D concentration after storage at 37°C for 1 month was less than 8%, the minimum change that could be detected. No significant change in physical characteristics of the immunoglobulin were detected.
The error of a single estimate of anti-D by the radioisotope method (¹²⁵I-labelled anti-IgG) used here was calculated to be such that the true value probably (p = 0.95) lay between 66 and 150% of the estimated value.     

Electrophoretic Precipitation and Radial Diffusion Methods for Assay of Anti-D Immunoglobulin Preparations

Abstract. The measurement of anti-D concentration has been carried out by two immunoprecipitation techniques utilizing sonicated D-antigen-containing stroma suspended in agarose. One method utilizes single radial immunodiffusion; in the other method, anti-D is moved into the agarose by electrophoresis. The results obtained form both immunoprecipitation techniques were compared to those obtained by a standard ¹²⁵I-antiglobulin method. The coefficient of variation of the difference between the results obtained by immunoprecipitation and those obtained from the ¹²⁵I-antiglobulin method were ±14% for the electrophoretic method and ±16% for the radial diffusion method.     

An Enzyme-Linked Immunosorbent Assay for the Quantitative Determination of Anti-D in Plasma Samples and Immunoglobulin Preparations

Here we present an enzyme-linked immunosorbent assay (ELISA) on microtiter plates for the quantitative determination of anti-D. This method is based on the solubilization of red blood cells sensitized with anti-D and the subsequent measurement of immunoglobulin G by ELISA. The measuring range of the assay is 40–5,000 IU/ml and the lowest quantifiable concentration in plasma is 0.5 IU/ml. The interassay relative standard deviation for concentrations above 130 IU/ml ranges from 3.2 to 8.1% and below 50 IU/ml from 10.5 to 19.7%. Comparison of ELISA with automated hemagglutination shows that the results of the two assays correlate well: r = 0.992, n = 26. The assay was validated for donor plasma samples and anti-D immunoglobulin preparations and it can also be used in assessing the severity of Rh (D) hemolytic disease during pregnancy.     

Accidental Injection of Anti-D Immunoglobulin to an Infant

Abstract. An Rh-positive infant, birthweight 2.6 kg, was inadvertently given an injection of 200 μg anti-D 5 h after birth. The rise in serum bilirubin concentration and the rate of fall of haemoglobin concentration suggested that a mild haemolytic syndrome was produced. No treatment was required and recovery was uneventful.     

A Comparative Study of Antiglobulin Antibodies and Residual Anti-D Between Recipients of Intramuscular Anti-D Immunoglobulin and Intravenous Plasma Anti-D1

Abstract. The incidence of ‘specific’ and ‘non-specific’ antiglobulin antibodies was determined among 648 multiparous females, 213 recipients of intramuscular anti-D immunoglobulin and 221 recipients of intravenous plasma anti-D. Results obtained six months after the administration of anti-D showed that the formation of ‘specific’ anti-Gm or anti-Inv was no greater in recipients of anti-D immunoglobulin or plasma anti-D than in the controls. The incidence of ‘non-specific’ antiglobulins increased from an expected 5% among recipients of intravenous plasma anti-D to almost 25% in mothers given intramuscular anti-D immunoglobulin. It is suggested that the raised ‘non-specific’ antiglobulins may be provoked by a residue of aggregated γ-globulin components which is known to be present in preparations of Cohn fraction II. Six months after the passive administration of Rh antibodies residual anti-D activity was more often observed in recipients of intramuscular anti-D immunoglobulin than in recipients of plasma anti-D.     

Immunoglobulin Preparations for HIV-Infected Patients

Patients with the acquired immunodeficiency syndrome (AIDS) suffer from a deficiency of cellular immunity. However, some HIV-infected children and adults suffer from recurrent upper respiratory tract infections suggestive of a failure to synthesise specific antibodies, despite the hypergammaglobulinaemia that is present. Intravenous immunoglobulin (IV IgG) appears to benefit HIV-infected children with recurrent infections, in doses similar to those used for treating patients with primary hypogammaglobulinaemia. In some HIV-infected adults, IV IgG also appears to reduce bacterial respiratory infections but the treatment schedules remain to be defined. In patients with life-threatening bleeding due to immune thrombocytopenic purpura associated with HIV infection, high dose IV IgG treatment (1-2 g/kg) also increases platelet counts but unlike other therapies for ITP, is not immunosuppressive and has no other serious adverse effects. It is likely that over the next few years, specific anti-HIV preparations will be evaluated. Neutralising antibody has been demonstrated in HIV-infected patients and a specific antibody preparation against HIV might either prevent HIV infection after initial exposure to the virus or slow the progression of HIV-related disease. However, the development of a specific, effective, neutralising anti-HIV immunoglobulin preparation (whether polyclonal or monoclonal) will require information about which HIV antigens elicit protective immunity and the role played by neutralising antibody in HIV-related disease.     

Fragmentation of Therapeutic Human Immunoglobulin Preparations

Some commercial batches of human therapeutic immunoglobulins (Ig) have been found to show evidence of molecular fragmentation when examined by molecular sizing methodologies including sodium dodecyl sulphate polyacrylamide gel electrophoresis [SDS-PAGE] and size exclusion high performance liquid chromatography (SE-HPLC). These batches all demonstrated impaired immunobiological activity (efficacy) as assessed by Fc function measured using a rubella haemolytic assay and as such are likely to be subpotent for therapeutic use. Fragmented Igs were characterized by the presence of at least three protein bands and peaks additional to monomeric IgG. Incubation of Igs with blood enzymes (plasmin and kallikrein) reproduced the fragmentation patterns observed for intrinsically degraded batches, suggesting that fragmentation occurred by contamination with these proteases from the source material (human blood) during manufacture. Intravenous Igs (IVIG) were found to be more susceptible to proteolysis than intramuscular Igs, probably as a consequence of the post-fractionation processing that some IVIGs receive which may induce molecular alterations, allowing enzyme access and fragmentation. Two of the products examined were found to be relatively resistant to proteolysis and both were formulated by processes that limit enzyme activity. These processes were inclusion of an enzyme inhibitor, α₂-macroglobulin, and formulation at acidic pH. Enzyme carry-over into the final product is a likely cause of Ig fragmentation, and reduction in levels of such contamination should lead to improvements in product stability and efficacy.     

Immunoglobulin G Subclass Distribution in Three Human Intravenous Immunoglobulin Preparations

In immunodeficiency patients the lack of immunoglobulins (Ig) can be total or partial with a specific IgG subclass imbalance masked by normal values for total IgG. In the latter case therapy with intravenous IgG preparations (IVIG) is generally beneficial, provided the IVIG preparations used originate from large pools of normal blood donors and exhibit a normal IgG subclass distribution. We have analyzed the subclass distribution of three IVIG products: Sandoglobulin (SAGL), GamimuneN (GI), Gammagard (GG), 6-10 lots each, in four different laboratories. The competitive enzyme immunoassays and radial immunodiffusion methods used different monoclonal and polyclonal antibodies specific for IgG1, IgG2, IgG3, and IgG4, respectively. Despite minor interlaboratory differences, the results show that the slightly lower IgG1 content of SAGL versus GI and GG was quantitatively compensated by a higher proportion of IgG2, that no differences existed in IgG3 levels, but that one preparation (SAGL) contained 2-3% of IgG4 compared to 0.5-1.5% in GI and below 0.5% in GG. This difference was significant, the two latter preparations being at or below the lower limit of what are considered to be normal values found in human adults. Such differences may have important clinical consequences.     

Radioelectroimmunoassay of Anti-D Immunoglobulin on Agarose Plates Containing Whole Red Cells

Abstract. By incorporating whole Rh(+) red cells in an agarose plate and adding a small amount of purified ¹²⁵I anti-D immunoglobulin as a tracer to standards and samples, a radioelectroimmunoassay (REIA) of anti-D immunoglobulin was possible. The results by this method agree with those obtained by the direct method.
The coefficient of variation (within plate precision) is less than 9% and the reproducibility is 12% (coefficient of variation for a plasma sample assayed on six occasions during 1 month).
The International anti-D serum was compared with the WHO standard preparation 68/419 on five occasions and found to contain 11; 11; 10; 9 and 11 μg per ampoule.     

Various Immunoglobulin Preparations for Intravenous Use

Abstract. In recent years, a number of intraveous immunoglobulin (IVIG) preparations have become available, primarily in Europe. Since these preparations differ in their physical, chemical and biologic properties, the physician is frequently presented with difficulties in selecting an appropriate product. Until now, in vitro studies have been the only reliable source of evaluating these preparations. This paper describes the commercially available IVIG preparations and assesses the properties of each.     

Failure of Anti-D Immunoglobulin to Remove Fetal Red Cells from Maternal Circulation

A Rhesus negative female was found post delivery to have circulating cells of fetal origin. The neonate was typed as D-positive. In spite of more than conventionally adequate doses of anti-D immunoglobulin given to the mother, the fetal cell count in the maternal circulation remained unchanged. Further investigation showed the fetus to be a Du variant and to exhibit a diminished reaction with the batch of anti-D immunoglobulin used.     

Anti-Rh D Activity of Commercial Intravenous Immunoglobulin Preparations

Background and Objectives: The presence of antibodies against Rh D in intravenous immunoglobulin (IVIG) products has been proposed as causing adverse reactions and blood grouping problems, although data on the incidence and titre of such antibodies is sketchy. This study was conducted to investigate methodology for carrying out haemagglutination tests on IVIG products, and to examine in retrospect 200 IVIG batches for the presence of anti-D and other haemagglutinins. Materials and Methods: Batches of commercial IVIG products were tested using the low-ionic-strength indirect antiglobulin test (LIAT). Positive batches were futher investigated using a typed red cell panel and direct haemagglutination tests. Results: 44 batches of IVIG gave positive LIAT. Of these, 12 contained specific anti-D. 11 batches had LIAT anti-D titres of 2–8; a further batch contained anti-D with a LIAT titre of 64. The remaining 32 batches agglutinated all erythrocyte phenotypes tested with LIAT titres ≤4; 14 of these batches showed positive direct haemagglutination and the presence of rouleaux. Conclusion: 6% of commercial IVIG batches contained specific anti-D, including one batch (0.5%) showing unusually high titre. 16% of batches contained haemagglutinins of unknown/non-specificity.     

Relative Functional Binding Activity of IgGl and IgG3 Anti-D in IgG Preparations

The intention to replace polyclonal IgG anti-D with human monoclonal antibody in the prophylaxis of haemolytic disease of the newborn requires knowledge concerning the relative content of IgG1 and IgG3 anti-D in prophylactic IgG preparations that are in present use. This has been carried out using a functional assay in which the absolute amount of IgG1 and IgG3 anti-D present on red cells was determined after incubation with IgG preparations. The assay was carried out by flow cytometry on 17 samples; expressed as a percentage of the total, the average value for the amount of IgG3 anti-D on the cells was 8% (range 1-18%). Similar measurements were also made on the anti-D present in 18 samples of antisera; IgG3 anti-D formed a larger fraction of the total, the average value being 17% (range 0-60%) confirming previously reported estimates. It is suggested that some of the low values found for IgG3 in IgG preparations may be due do preferential loss during production.     

Opsonic and Physicochemical Characteristics of Intravenous Immunoglobulin Preparations

The composition and opsonizing activity of five commercially available immunoglobulin preparations for intravenous use (Venoglobulin I, Venilon, Gammagard, Polyglobin, and Sandoglobulin) were studied. The composition of these preparations does not differ very much as far as total protein, immunoglobulin class and IgG subclass concentrations are concerned. The only exceptions were that Veniglobulin I, Gammagard and Sandoglobulin contain IgA, which might cause side effects in patients with anti-IgA antibodies, Gammagard contains very little IgG4, and Venilon and Polyglobin contain no and almost no IgG3, respectively, which might explain their very low opsonic activity. It was found that Venilon and Gammagard activate complement in the ready-for-infusion state. The opsonic activity of Venoglobulin I, Sandoglobulin and Gammagard is about equal to that of inactivated serum: Staphylococcus aureus, Escherichia coli with K antigen, Streptococcus pyogenes and Streptococcus group B are well opsonized and E. coli without K antigen and Streptococcus pneumoniae are poorly opsonized.     

A Second Outbreak of Hepatitis C Virus Infection from Anti-D Immunoglobulin in Ireland

OBJECTIVE: To investigate the infectivity for hepatitis C virus (HCV) of intravenous anti-D immunoglobulin batches manufactured in Ireland between 1991 and 1994. METHODS: Women who had received anti-D manufactured between 1991 and 1994 were screened for serological markers of HCV infection and for the presence of HCV RNA by RT-PCR amplification and virus genotyping. RESULTS: 44 women exposed to anti-D manufactured between 1991 and 1994 were polymerase chain reaction positive for HCV RNA, 19 of whom were infected with genotype 3a virus shown by phylogenetic analysis of the NS5B gene to be closely related to that from the single implicated donor. CONCLUSIONS: Anti-D manufactured in 1991-1994 transmitted infection of HCV genotype 3a. The prevalence of HCV-specific antibody in anti-D recipients was relatively low (0.59%), consistent with the low level of virus RNA in these anti-D batches.     

Radio-Immunoassay of the Functional Activity of Anti-D (Rh) Preparations using a Human Monoclonal 125I-Labelled Anti-D

Abstract. The prophylactic effect of anti-D in the prevention of maternal immunization to the D antigen of the Rh system depends in the first instance on its ability to bind to fetal Rh-positive red cells. The functional activity of the injected anti-D in this respect is dependent on both the plasma concentration and functional affinity constant of the antibody, and both factors should be taken into account in assessing IgG anti-D preparations for clinical use. A radio-immunoassay utilizing ¹²⁵-I-labelled human monoclonal anti-D has been developed which measures the amount of antibody bound to red cells after the addition of an aliquot of the test anti-D preparation. The assay is thus a functional test in that it measures the ability of the anti-D to react with the antigen. The functional activity of the test anti-D is compared to that of the international standard, and the concentration is then expressed in terms of μg-equivalents, that is, its equivalence in functional terms to the stated amount of international standard. The radio-immunoassay was compared to the currently used agglutination assay, and the results were found to give good agreement in 18 out of the 21 samples assayed.