Nicholas J. Marshall

An Investigation into the Lability of the Bioactivity of Human Growth Hormone Using the ESTA Bioassay

We compared the bioactivity attributable to human growth hormone (hGH) in serum samples, determined at the time of their collection, with that after storage for 2-18 months at -20 degrees C. The samples were obtained from volunteers and patients who underwent provocative tests of hGH secretion, and the bioactivity was determined in the ESTA bioassay, which is based upon the use of Nb2 cells. We report that, in some subjects, the bioactivity of samples collected at the response peaks deteriorated on storage for as little as 2 months. The decrease in hGH bioactivity was systematic in that it consistently declined so as to approach the values initially determined by an immunoassay (Hybritech IRMA). This differential lability was a characteristic of the peak samples, and was not observed for either samples collected before and after the peaks of hGH secretion or for purified preparations of hGH which were subjected to a range of freeze/thaw and storage regimens. We suggest that this unusual lability is indicative of transient shifts in the spectrum of the variants of hGH which are present in the circulation following stimulation by provocative agents. This study emphasises the need to minimise the risk of introducing storage artefacts in investigations into the responses of hGH to provocation.

The mechanism of action of interferon-α (IFN-α) in hairy-cell leukaemia; Hu-IFN-α2 receptor expression by hairy cells and other normal and leukaemic cell types

To investigate the possible direct effects of interferon-α (IFN-α) in hairy-cell leukaemia, IFN-α receptor expression by hairy cells (HCs) (11 cases) was measured by a radiolabelling technique and compared with that of MOLT-4, chronic lymphocytic leukaemia (CLL; 14 cases) and various other leukaemic and normal cell types. Purified peripheral blood (PB) and splenic HCs showed higher levels of receptor expression (approx. 1000 ± 200 binding sites/cell; 11 cases tested) than other normal and leukaemic cells types. Purified normal PB and tonsil B cells showed low levels of receptors (approx. 120 ± 100 binding sites/cell), while a range of B-cell leukaemias displayed intermediate levels of expression (approx. 100–500 sites/cell). In the 15 cases of CLL tested, 530 ± 330 binding sites/cell were demonstrated, the high standard deviation reflecting the fact that approximately one third of cases had receptor levels comparable with those in HCL. Normal and HCL T cells, red cells and platelets had no demonstrable IFN receptors. It is suggested that these findings may be relevant to the efficacy of IFN in hairy-cell leukaemia.

An improved metaphase index assay for detecting thyroid growth stimulators using FRTL-5 thyroid cells cultured on a microtitre plate

The metaphase index assay (MIA) for thyroid growth stimulators, as originally described, used FRTL-5 thyroid cells cultured in Bellco culture chambers and glass microscope slides. The metaphases were observed using the nuclear strain aceto-lacto orcein. However the surface properties of the glass proved to be variable and so polystyrene microscope slides were substituted. The aceto-lacto orcein stain was found to be unsuitable for use with polystyrene because of the solvents and mountant used. Therefore combinations of various other nuclear stains and mounting media were tested. The Giemsa stain, which was found to be the most satisfactory, could be applied to FRTL-5 cells maintained on the large variety of plastic supports now available for tissue culture, e.g., 96 well microtitre plates. This permitted the design of an MIA which is much more convenient, robust and economical in its use of clinical samples. The results with seven IgG preparations derived from the sera of patients with a variety of thyroid disorders are presented. In its revised form, the metaphase index assay provides a rapid screening assay for thyroid growth stimulators, such as autoantibodies and TSH.