A female patient was born without any perinatal problems and her psychomotor and growth development had been normal. Her father had Crohn’s disease and her elder brother had atopic dermatitis. She developed allergic conjunctivitis when she was around 10 years old and was administered betamethasone eye-drops. She also developed a butterfly rash and photosensitivity at the age of 14. And then, she suddenly developed a left visual disturbance without any premonitory sign at the age of 15. Her visual acuity (left: 0.04, right: 1.2) showed asymmetry. Her eye fundus examination showed massive left intraretinal hemorrhaging due to CRVO (Figure 1). Her blood pressure was 100/70 mmHg and she had not history of any cardio-vascular disorders or diabetes.
Her laboratory findings showed pancytopenia with a WBC count of 4,680/mm3, RBC count of 351 × 104/mm3, Hb level of 9.9 g/dl, Ht level of 30.3% and PLT count of 16.1 × 104/mm3, an increased erythrocyte sedimentation rate of 54 mm/hr (<10), but APTT of 28.0 seconds was within the normal range. There was increased anti-nuclear antibody titer of x640 (<x40), ds-DNA antibody titer of 377.7 IU/ml (<12.0) and ss-DNA antibody titer of 722.4 AU/ml (<25.0) and a decline in complement levels of C3 14 mg/dl (86–160), C4 3 mg/dl (17–45) and CH50 < 10 U/ml (23–46). However, both her anti-CL IgG antibody of 9 U/ml (<10; EIA method), anti-β2 glycoprotein I antibody of 1.3 U/ml (<3.5; EIA method) and lupus anticoagulant of 1.2 (<1.4; Dilute Russell’s Viper Venom Time) were within the normal range.
Intravenous low-molecular weight heparin of reviparin was initiated, and she received steroid pulse therapy, oral fexofenadine and levocabastine eye-drops. The intraretinal hemorrhaging gradually improved, and the patient was treated with warfarin, aspirin, prednisolone and tacrolimus. Her anti-CL IgG antibody titer was examined four times every two weeks, however, all of evaluations showed negative results (2/3/1/1 U/ml). The second examination of the anti-β2 glycoprotein I antibody titer also showed a level less than 1.3 U/ml.
After informed consent was provided by the patient and parents, we examined the patient for other antiphospholipid antibodies according to the Guidelines of the European Consensus described the development of the antiphospholipid IgG antibody ELISA assay, as described previously [3]. Briefly, 1 ml of whole blood was drawn twice at an interval of 12 weeks. Microtitre plates were coated with 50 μg/ml phosphatidylcholine (PC, Nacalai tesque, Kyoto, Japan), phosphatidylethanolamine (PE, Sigma, St. Louis, USA), phosphatidylinositol (PI, Nacalai tesque, Kyoto, Japan) and phosphatidylserine (PS, ChromaDex, Inc, California, USA) and were incubated overnight. On the following day the plates were incubated with triplicate serum at a 1/10 dilution for one hour. After wash, the plates were incubated with goat anti-human IgG (Santa Cruz Biotechnology, Inc, California, USA) at a 1/2,000 dilution for one hour. Using substrate of o-phenylenediamine (Sigma, St. Louis, USA), the absorbance was read at 450 nm with ELISA reader. Age-matched three patients with APS with anti-CL IgG antibody, four patients with SLE and eight patients with non-collagen disease controls were also enrolled as disease controls. The positive cut-off value was defined as two standard deviations above the mean in the controls subjects. Since there were no positive control antiphospholipid antibodies, we calculated the optical density (OD) according to the method used in previous reports [3].
Her anti-PC IgG antibody (0.342 OD), anti-PE IgG antibody (0.190 OD), anti-PI IgG antibody (0.290 OD), and anti-PS IgG antibody (0.214 OD) were increased in comparison to not only normal controls (anti-PC; 0.095 ± 0.009 OD, anti-PE; 0.079 ± 0.019 OD, anti-PI; 0.082 ± 0.012 OD, anti-PS; 0.084 ± 0.033 OD) but also other patients with APS (anti-PC; 0.181 ± 0.076 OD, anti-PE; 0.117 ± 0.021 OD, anti-PI; 0.130 ± 0.039 OD, anti-PS; 0.129 ± 0.094 OD) and SLE (anti-PC; 0.117 ± 0.019 OD, anti-PE; 0.110 ± 0.038 OD), anti-PI; 0.097 ± 0.046 OD, anti-PS; 0.090 ± 0.037 OD). The elevation of anti-PC (0.202 OD), anti-PE (0.114 OD), anti-PI (0.181 OD), and anti-PS (0.139 OD) IgG antibodies persisted after 12 weeks.