antigen, and platelet aggregation by ristocetin exclude von Wiflebrand’s disease. Factor-viii activity and antigen levels in both parents and the patient’s brother were normal. This suggests that her mother is not a carrier of classical haemophilia. Chromosome analysis of peripheral-blood leucocytes showed normal female karyotype (46, XX). Classical haemophilia in this girl with two X chromosomes seems to be a sporadic case derived from normal parents. It is not clear whether she is a homozygous haemophiliac or a heterozygous carrier. However, the chance of spontaneous mutation in both X chromosomes is much smaller than that in one X chromosome. Although the factor-vm activity of 7% is unusually low for carriers, this could be explained by the Lyon hypothesis,’ if we assume that most normal X chromosomes in the XX cells have been inactivated. 1st Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan
T. KAMIYA K. KOIE J. ISHIGURO K. OGATA
T4 RADIOIMMUNOASSAY AND THYROXINE-BINDING GLOBULIN CONCENTRATION
SIR,-We have noticed that, in patients with high serum concentrations of thyroxine-binding globulin (T.B.G.) due to pregnancy, oral contraceptives, or hereditary elevation of T.B.G., the serum-thyroxine (T4) varies strikingly, depending on the assay system used. When the T.B.G. concentration was high, T4 radioimmunoassay (R.!.A.) using a resin to separate antibody-bound from free T41 gave consistently lower values than either competitive protein binding using the Murphy-Pattee method2 or R.I.A. using an immobilised antibody. Ethanol extraction of serum before assay resulted in higher T4 values with the resinseparation method, but not with the other methods. Most R.I.A. techniques for T4 include an inhibitor of T4-T.B.G. binding, and we use 8-anilino-l-naphthalene sulphonic acid at a final dilution of 0.9g/1. This is a higher concentration of inhibitor than is used in most other reported T4 R.I.A.s,’ 34 nevertheless it seemed probable that T.B.G. was being incompletely blocked and was competing for the ’Z’I-T4 label. The resin removes mainly free T4, so that label bound to T.B.G. would be included with the antibody-bound fraction, leading to a falsely low T4 concentration. To test this explanation, we studied the effect of prior dilution of serum (with T4-free serum) on the resin-separation T4 R.I.A. results in serum from ten thyrotoxic patients, seven oralcontraceptive users, ten pregnant women, and fifteen individuals with congenital T.B.G. elevation. We have also examined the effect of increasing the concentration by a factor of three in the group with congenital T.B.G. inhibitor elevation. Dilution of thyrotoxic serum had no effect on T4 values when corrected for dilution, and only a slight effect was noted in serum from oral-contraceptive users (see figure). The T4 in pregnancy serum rose from a mean (+ S.E.M.) of 121+4.0 nmot/1 in undiluted serum to 143±7.1 nmol/1 after four-fold dilution. In hereditary T.B.G. elevation there was an even more striking increase, from 177±10-5 to 236±27.0 nmol/1. Increasing the inhibitor concentration raised the mean T4 (± s.E.M.) to 203 ±24-0 nmol/1 in this group (see figure). These results support the suggestion that in pregnancy and other high T.B.G. sera, blocking of T.B.G. by 8-anilino-l-naph7
Lyon, M. F. A. Rev. Genet. 1968, 2, 31. 1. Ratcliffe, W. A., Ratcliffe, J. G., McBride, A. D., Harland, W. A., T. W. Clin. Endocr. 1974, 3, 481. 2. Murphy, B. E. P. J. Lab. clin. Med. 1965, 66, 161. 3 Chopra, I. J. J. clin. Endocr. Metab. 1972, 34, 938. 4 Mitsuma, T., Colucci, J., Shenkman, L., Hollander, C. S. Biochem. Res. Commun. 1972, 46, 2107.
Effect of dilution and raising inhibitor (ANS) concentration on T4 measurement by resin-separation R.I.A. from P