SUMMARY Evidence for the presence of circulating immune complexes was found in thirtyfour out of fiftyfive samples from fortyseven patients with haemophilia A. In eleven patients the complexes, precipitated from the blood with polyethylene glycol, were digested with pepsin. The F(ab)2 antibody was tested, and found to have neutralizing activity against coagulant Factor VIII in two patients. In one of these no free antibody had ever been found in the plasma, while in the other the antibody was concentrated tenfold in the complex. In two other samples free without complexed antibody was found. In comparison, IgGcontaining complexes were found in nine out of nineteen patients with von Willebrands disease and no complexes were found in the sera from twelve multitransfused thalassaemics. PEG precipitation is a useful technique for the preparation of concentrated immune complexes for further study such as antigen identification.
Clin exp Immunol (1980) 39, 315-320 Circulating immune complexes containing anti-VIII antibodies in multi-transfused patients with haemophilia A M D KAZATCHKINE,* Y SULTAN, E J BURTON-KEE & J F MOWBRAY Department of Experimental Pathology, St Mary's Hospital Medical School, London, and Department of Haematology, Hopital Cochin, Paris, France (Accepted for publication July 1979) SUMMARY Evidence for the presence of circulating immune complexes was found in thirty-four out of fiftyfive samples from forty-seven patients with haemophilia A In eleven patients the complexes, precipitated from the blood with polyethylene glycol, were digested with pepsin The F(ab')2 antibody was tested, and found to have neutralizing activity against coagulant Factor VIII in two patients In one ofthese no free antibody had ever been found in the plasma, while in the other the antibody was concentrated tenfold in the complex In two other samples free without complexed antibody was found In comparison, IgG-containing complexes were found in nine out of nineteen patients with von Willebrand's disease and no complexes were found in the sera from twelve multi-transfused thalassaemics PEG precipitation is a useful technique for the preparation of concentrated immune complexes for further study such as antigen identification INTRODUCTION Acquired inhibitors of Factor VIII develop in 8-14% of patients with severe haemophilia A treated with plasma or anti-haemophilic Factor concentrates (Shapiro & Hultin, 1975) These inhibitors can be recognized by their specific ability to inactivate procoagulant activity of human Factor VIII (VIIIc) and have been identified in most series as oligoclonal IgG antibodies Recent in vitro studies using antiFactor VIII plasma, or semi-purified anti-VIIIc immunoglobulins have provided evidence for immune complex formation between anti-VIIIc and purified Factor VIII or Factor VIII from cryoprecipitates (Allain & Frommel, 1973; Lavergne, Meyer & Reisner, 1976; Lazarchick & Hoyer, 1977) Using a simple semi-quantitative test we have studied the occurrence of circulating immune complexes in vivo in multi-transfused patients with haemophilia A, and we have demonstrated the specificity of some of the complexed antibodies MATERIALS AND METHODS Forty-five citrated plasmas and ten sera were obtained from forty-seven patients with haemophilia A The ten sera were obtained from patients with the severe form of the disease (VIIIc below 0-01 iu/ml); of these, three (patients BP, BS and CP, Table 1) had a circulating inhibitor of Factor VIII, and one had a previously documented inhibitor but had no VIIIcneutralizing activity in the serum at the time of the study (patient LA) With the exception of two patients with circulating inhibitor (BS and CP), all patients received either therapeutically or prophylactically 1500 to 100,000 iu of Factor VIII, as cryoprecipitate, within twelve weeks of the study For comparison, plasma samples were obtained from nineteen patients with severe von Willebrand's disease who were regularly transfused with Factor VIII concentrate, sera were obtained from twelve multi-transfused patients with thalassaemia (sera kindly provided by Dr E Letsky, Institute ofChild Health, London), * Present address: Department of Medicine, Harvard Medical School and Robert B Brigham, Hospital, Boston, MA, USA Correspondence: Dr M Kazatchkine, The Seeley G Mudd Building, 250 Longwood Avenue, Boston MA 02115, USA 0099-9104/80/0200-0315 $02-00 () 1980 Blackwell Scientific Publications 315 316 M D Kazatchkine et al and thirty-eight plasmas and thirty-four sera were obtained from twenty-four healthy individuals, together with eight pools of serum from normal healthy subjects Plasmas were stored at - 20'C and - 70'C; sera were separated within hr aftdr clotting and stored at - 70'C in glass bottles until analysis Circulating immune complexes These were measured by a semi-quantitative assay using the precipitation of complexes from serum or plasma with 2% polyethylene glycol (PEG) followed by measurement of the amount of IgG and Clq in the precipitate For the test, 0-1 ml of a 12% solution of PEG 6000 (BDH Ltd., Poole) in veronal buffer containing 60 mM EDTA, pH 7-6, was added to ml of the test sample in a polystyrene tube After incubation for 18 hr at 4VC, the samples were centrifuged at 1000 g for 20 at 40C The supernatants were decanted and kept at 40C The precipitates were washed once in ml veronal buffer containing 2% PEG, 10 mm EDTA, pH 7-6, and redissolved in 0-5 ml of veronal buffer pH 7-6 The amount of IgG and Clq in the precipitate was determined by single radial immunodiffusion (SRID) using 1% agarose gels in veronal buffer containing 40 mm EDTA, pH 8-6, and appropriate dilutions of specific antiserum The values in eighty normal samples were 39+ 0-15% of serum IgG concentration (mean+ s.d.) for the precipitated IgG from ml serum and 44 ug± 20 (mean+ s.d.) for the precipitated Clq Values of precipitated IgG and Clq from the test samples which were more than standard deviations above the mean values obtained in the normal samples were regarded as positive The measurement of Clq in the starting serum and in the supernatant by SRID served as an internal control for Clq recovery The sensitivity of this assay allows the detection of 10 ,ug/ml of heat aggregated IgG IgA in the precipitates was detected by double diffusion in agarose plates and measured by rocket electrophoresis at v/cm overnight in 1% agarose gels containing 40 mm EDTA buffer, pH 8-6, and 2-5% v/v rabbit anti-human IgA antiserum F(ab')2 antibodies These were prepared from the PEG precipitates by pepsin digestion The precipitates obtained from ml of starting serum were adjusted to pH with N and 0-2 N acetic acid, and 5% pepsin by weight in acetic acid was added to give a final substrate/enzyme ratio of 20: The digestion was performed for hr at 37°C; the pH was raised to with N NaOH and then to with M phosphate buffer pH 8-0 After addition to rabbit hyperimmune serum used as a carrier, the F(ab')2 fragments were precipitated with 20% Na2SO4 at 37°C for 30 min, and tested for antibody activity after overnight dialysis against phosphate buffered saline (Lachmann, 1971; Dambuyant, Burton-Kee & Mowbray, 1978) Complement components and immunoglobulins Serum Clq, C3, C4, C5, IgG, IgA and IgM concentrations were measured by SRID in agarose using monospecific antisera raised in rabbits C2 functional activity was measured in an assay using the serum of a patient totally deficient in haemolytic and antigenic C2 as a substrate Factor B activity was measured as described (Martin et al., 1976) Factor VIII inhibitor activity This was measured as described in 1975 at a Bethesda conference (Kasper et al., 1975) Hepatitis B surface antigen (HBs) and antibody to HBs These were detected and titrated by inhibition of haenmagglutination and passive haemagglutination tests RESULTS Results obtained in patients with haemophilia A, von Willebrand's disease and transfusion-dependent thalassaemia are shown in Fig The amounts of 2% PEG precipitable IgG were significantly raised more than standard deviations above the normal mean in thirty-four of fifty-five samples from patients with haemophilia (thirty of forty-seven patients) (P