THE LANCET, OCTOBER
IN-VITRO BEATING OF HUMAN EMBRYONIC CARDIAC TISSUE SIR,-Piiproinen 1 reported that, with the ultrasound Doppler method, fetal heart activity could be detected as early as 44 days from the beginning of the last menstruation. Recently we have observed the beating of early-gestation human embryonic cardiac tissue in vitro. We should like to report our finding, which supplements Piiproinen's. A piece of tissue (1/2 mm. in size) from a spontaneously aborted embryo (63 days from last menstrual period) was studied for cytogenetic reasons. The tissue was cultured in minimal essential medium supplemented with 15% fetal
UNUSUAL ECTODERMAL DYSPLASIA SIR,-For the past two years we have followed two brothers, aged 5 and 3 years, with an unusual form of ectodermal dysplasia (E.D.). This condition is characterised by sparse, short, fragile scalp hair, mild hypoplasia of finger and toenails, but normal eyebrows, eyelashes, and teeth. There is also hypoplasia of the dermal ridges and severe hypohidrosis. In each boy a lace-like hyperpig, mentation developed in the first few weeks of life and became more intense during the next three years, and in the older one has begun to fade during the past year. Both boys are of normal physical and mental development. A 4-year-old sister is normal. On careful skin biopsy in both boys, unusual numbers of mast cells were found infiltrating the dermis, together with increased numbers of melanophores. The combination of mastocytosis (M) and this form of E.D. in otherwise normal children is unusual. Detailed histological and biochemical studies of this M.E.D. syndrome will be published. Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada. Regional Hospital, University of Linkoping, Sweden.
Electron lDicrograph of a partial beating cell showing lDyofibrils (MF) with Z line (Z), intercalated disc (insert), and lipid droplets (L).
This cell is from a human embryonic tissue-culture 26 days old. Main photograph and insert reduced by half, from x 22 and x 37, respectively.
calf serum and antibiotics in a 25 em. Falcon flask. On the fourth day, rhythmic beating of the explanted tissue and the surrounding cells was observed. The beating was documented with a Leicina movie camera attached to the Leitz phase-contrast inverted microscope. The rate of beating was about 10 per minute and fluctuated between 5 and 12 per minute until the 10th day, when the tissue broke into three fragments. One fragment was floating in the medium and was removed for electron microscopic study; the other two fragments remained attached to the flask, and each maintained its own beating. On the 26th day of culture, beating in both the fragments became slow and weak. The cells were then sacrificed for E.M. examination. Myofibrils with Z line, intercalated disc, and lipid droplets were seen in the electron micrographs (see accompanying figure). Since all these cellular components are ultrastructurally characteristic of human embryonic myocardinum in vivo 2 and beating rat heart-cells in vitro," we conclude that the explant in this study was cardiac tissue. Detailed description of the findings will be published elsewhere. We thank Dr Godfrey P. Oakley for supplying the tissue. Cellular Genetics Laboratory, Pathology Division, Center for Disease Control, Atlanta, Georgia 30333, U.S.A.
ANDREW T. L. CHEN SUZANNE TRUSLER CAREY S. CALLAWAY
1. Piiproinen, O. Lancet, 1973, u, 508. 2. Leak, L. V., Burke, J. F. Anat. Rec. 1964, 1"9,623. 3. Cedergren, B., Harary, I.J. Ultrastruct, Res. 1964, 11,428.
C3 STANDARDS? SIR,-We agree with Dr Vladutiu (April 26, p. 979) when he says that C3 standardisation is important not only in immunological but also in clinical laboratories. In 1972 a World Health Organisation subcommittee on complement standardisation was formed under the chairmanship of Dr Leo Van Es of the Netherlands Red Cross BloodTransfusion Service in Amsterdam. This committee is conducting multi-laboratory trials of the stability of complement components, comparing complement measurements, and evaluating methods for measuring the activity and protein concentration of complement components. During a session of this standardisation subcommittee at the European Complement Workshop in 1974, we briefly reported our findings on immunochemical C3 measurement, and some of our results have also been published.v ! We compared aliquots of sera on the same plate using different anti-C3 sera. One aliquot was kept at -70°C to -100°C to inhibit C3 conversion, while the other aliquot of the same serum was incubated under appropriate conditions at +37°C for 3-7 days for conversion of C3 in the sample. The mean differences between the values of the corresponding fresh and aged aliquots of the same sera ranged from plus 93% to minus 17% of the fresh aliquot value, depending on the anti-C3 serum used. This variation in the differences must be attributed to the varying specificity of the different anti-C3 sera for the antigenic determinants of fresh and converted C3. For this reason the stage of C3 conversion reached is critical to the imrnunochemical measurement of C3. The change in C3 value due to C3 conversion depends on the quality of the anti-C3 serum, which may vary and is not predictable. This finding contrasts with the results of Davis et al. 5 who proposed the use of a correction factor for comparing fresh and aged serum values. C3 should be measured immunochemically either as I. Spath, P., Gabl, F. Blue, 1974,28,224. 2. Spath, P., Gabl, F. C/in. Res. 1974,22, 430A. 3. Spath, P., Gabl, F. Protides bioi. Fluids, 1975,22,551. 4. Spath, P. in Der heutige Stand der Rheumatologie in Forschung und Praxis (edited by S. Sailer and E. Borkenstein); p. 116. Vienna, 1974. 5. Davis, N. c., West, C. D., Ho, M. C/in. Chern. 1972, 18, 1485.
THE LANCET, OCTOBER
~IC or ~IA protein. Edetic acid/plasma is suitable for the measurement of ~IC since it inhibits C3 conversion, which may occur very rapidly in some sera at room temperature.' The disadvantage of measuring ~lC is that no commercial ~1 C standards are available. On the other hand measuring C3 as ~IA protein seems more convenient because ~IA standards are available. However, since the complete conversion of C3 in serum may take 7 days at +37°C, measuring ~IA accurately is a relatively time-consuming procedure. In-vitro acceleration of C3 conversion could be very helpful, but changes of the protein during such a procedure may introduce a further error, as Kohler and MullerEberhard' found using hydrazine and as we found using thrombin. We are currently investigating this aspect of C3 measurement. In contrast to the findings of Davis et al. 6 we did not observe significant differences between plastic and glass tubes. This could be due to the kind of material used. In accord with Laurell 8 we found significant differences between values obtained by the single radial immunodiffusion and the electroimmunodiffusion method, even when using the same reagents. These pitfalls of the immunochemical measurement of C3 must be borne in mind, especially by those laboratories using commercially produced immunodiffusion kits.
2 Medizinische Abteilung, Landeskrankenhaus, A-8036 Graz, Austria. Abteilung fur med. chern. Labordiagnostik, I Med. Univ. Klinik, Spitalgasse 23, A-L090 Vienna, Austria.
STEROID THERAPY AND THE ADRENALS SIR,-While agreeing with your leader (Sept. 20, p. 537) that adrenal atrophy is inevitable after prolonged corticosteroid administration, related to dose and duration (presumably a continuous regimen), we have observed remarkable resilience of the hypothalamo-pituitary-adrenal (H.P.A.) axis to high-dose corticosteroid therapy given intermittently. Prednisolone is now widely used in high-dosage intermittent regimens in the combination chemotherapy of myeloma" and Hodgkin's disease. 10 In studying 6 patients with myeloma and 4 patients with Hodgkin's disease who received intermittent high-dose prednisolone either with melphalan (in myeloma patients) or with vinblastine, mustine, and procarbazine (in the Hodgkin's group), we performed standard insulin hypoglycemia tests (I.H.T.) 36 hours after the last dose of prednisolone at the conclusion of a course of therapy. The object was to assess the response of the H.P.A. axis to stress in the absence of steroid therapy and thus to predict requirements for additional steroids in the event of severe intercurrent illness, particularly infection and hsemorrhage. Full details will be published shortly. In the myeloma group, 2 patients had their steroid dosage reduced over 5 days after their 19th and 12th courses of treatment and had normal plasma-corticosteroid responses in the I.H.T. Of 4 patients whose steroid therapy was stopped 6. Laurell, A.-B. Personal communication. 7. Kohler, P. F., Muller-Bberhard, H. J. J. Immun. 1967, 99, 1211. 8. Laurell, A.-B. Meeting of the W.H.O. Subcommittee on Complement Standardisation, European Complement Workshop, Heidelberg, 1974. 9. Alexanian, R., Haut, A., Khan, A. U., Lane, M" McKelvey, E. M., Migliore, 1'. J., Stuckey. W. J., Wilson, H. E. J. Am. med. Ass. 1969,208, 1680. 10. De Vita, V. '1'., Jr., Serpick, A. A., Carbone, P. P. Ann. intern. Mcd. 197U, 73,881.
abruptly at the end of the week's course, there were 3 normal and I abnormal responses, following 18, 13, 19, and 29 courses respectively. The patient with an abnormal insulin test exhibited a normal plasma-corticotrophin response to hypoglycemia, indicating that suppression had occurred at the adrenal level. In the Hodgkin's group all steroid therapy was stopped abruptly, and I.H.T.S were normal in 3 cases (who had 8, 12, and 5 courses of combination chemotherapy) and abnormal in 1 case (who had 6 courses). This last patient had received 40 mg prednisolone daily in courses of 1 week, whereas the other 3 patients had courses of 2 weeks. His plasma-corticosteroid response to intravenous tetracosactrin 250 [lg was poor, again suggesting suppression at the adrenal level. Intermittent oral steroid therapy alone has been shown to suppress the H.P.A. axis when given 3 days per week in mean dosages of15·5 mg of prednisolone per day;'! but when steroids are given on alternate days for a shorter period of time normal H.P.A. function has been demonstrated.P The integrity of the H.P.A. axis has been remarkably preserved in most of the patients treated with combination chemotherapy whom we have studied, but suppression can definitely occur. With the development of finite treatment regimens in myeloma and lymphomas, we feel that full assessment of the H.P.A. axis by tetracosactrin stimulation (and, if necessary, insulin hypoglycemia tests) is advisable on completion of chemotherapy. University Departments of Medicine and Clinical Chemistry and Department of Hematology, Royal Infirmary, Edinburgh.
K. S. WILSON
J. SETH A. C. PARKER
11. Malone, D. N. S., Grant, I. W. B., Percy-Robb, I. W. Lancet, 1970, ii, 733. 12. Jacobson, M. E. Postgrad. Med. 1971,49,181.
Obituary ERNEST ARTHUR FREEMAN M.B. Lond., F.R.C.S.
By the death of Mr E. A. Freeman on Sept. 14, at the age of 74, Wolverhampton has lost one of its prominent personalities and an influential figure in the orthopedic affairs of the West Midlands. Born in Streatham in 1900, he received his secondary education at Westminster City School and in the last weeks of the 1914-18 war was called up to serve in the Royal West Surreys. This enabled him to gain an ex-Serviceman's education grant which took him to St. Bartholomew's Hospital, where he proved outstanding. Coming to the Royal Hospital, Wolverhampton, in 1931, he directed his enormous energy and enthusiasm to setting up what soon became an overwhelmingly busy fracture department. At his manipulation sessions he would put even the most massive man, under general anesthesia, through the full range of spinal movement with effortless ease. In 1940, in the treatment of war casualties, he became associated with Patshull, which through his constant endeavours became a very active rehabilitation centre and following his stimulus has continued to help the injured and disabled to the present time. Later when convoys of wounded began to arrive he branched out from the hard-pressed facilities at the Royal Hospital to Wordsley Hospital, near Stourbridge, where he formed an ever enlarging orthopedic unit which became the catalyst for development of all the other surgical activities of the hospital. The massive amount of work he was able to carry out depended on the elimination of "dead" time. Secretary and