LABELLED ANTIBODIES FOR TUMOUR LOCALISATION

367

Satisfactory levels rejection episodes. This

case

were

illustrates

maintained thereafter with

no

further

’

a

potentially lethal

combination of drug

treatment. It appears that the combination of intravenous

trimethoprim therapy resulted in a marked cyclosporin levels so that severe rejection occurred in a patient who was previously stable. Oral medication did not appear to have a similar effect. The with these ua drugs -mechanism is not known. J. WALLWORK

sulphadimidine reduction in

and

serum

W.llU l.U",","B’"

aam

Cardiothoracic Surgical Unit,

Papworth Hospital, Papworth Everard, Cambridge CB3 8RE

C. G. A. MCGREGOR F. C. WELLS R. CORY-PEARCE T. A. H. ENGLISH

TUBERCULOSTEARIC ACID AS A DIAGNOSTIC MARKER IN TUBERCULOUS MENINGITIS

SIR,-Ever since Robert Koch discovered the tubercle bacillus one hundred years ago, the diagnosis of tuberculosis has been a timeconsuming procedure. We have reported the use of gas chromatography/mass spectrometry (GC/MS) in the rapid diagnosis of pulmonary tuberculosis by analysing sputum for compounds of mycobacterial origin. A 24-year-old male was referred to the department of infectious diseases, University Hospital, Copenhagen, on April 18, 1982, with mental confusion and drowsiness, stiffness of the neck, and an increased body temperature. Cerebrospinal fluid (CSF) samples showed lymphocytic meningitis; 253 white cells/1 (62% polymorphonuclear cells). The CSF glucose was 0 -9 mmol/1 and the plasma glucose was50mmol/1. The protein content of the CSF was 2-71 mg/ml. At first listeria meningitis was suspected and ampicillin was instituted. The following day tuberculosis meningitis was thought to be a more likely diagnosis, and rifampicin, myambutol, and isoniazid treatment was started. On April 22 acid-fast bacilli were detected in the sputum. On April 28, mycobacteria were detected in cultures of CSF by microscopy of the medium and in smears of such specimens (from a sample collected on April 18). Later also cultures of urine showed

’

Scan number

diagnostic battery available in tuberculous meningitis and has the obvious advantage that a result can be obtained within one or two days. The GC/MS technique used is very sensitive and can detect tuberculostearic acid in picogram (10-12g) amount.3 To the best of our knowledge this is the first demonstration of tuberculostearic acid in the CSF of a patient with tuberculous

meningitis. Institute of Medical S-223 62

The CSF sample in which tuberculostearic acid was detected by GC/MS had been collected on April 19, before antibiotic treatment was

instituted.

The figure shows the mass chromatogram obtained by analysing a CSF sample taken on April 18. The peak area of methyl tuberculostearate corresponds to about 50 pg of tuberculostearic acid, which means a concentration of about 3 ng/mg CSF. The patient’s Mantoux reaction, which on admission was negative, had become positive 5 weeks later. He continued his treatment with myambutol and isoniazid after being discharged July 16. He improved slowly, and when seen on Nov. 23 his general condition was good. Tuberculostearic acid is a characteristic constituent of mycobacteria and certain other members of the actinomycetales family.2 The demonstration of this acid by GC/MS adds to the 1. Odham G, Larsson L, Mårdh P-A. Demonstration of tuberculostearic acid in sputum from patients with pulmonary tuberculosis by selected ion monitoring. J Clin Invest 1979, 63: 813-19. 2. Larsson L, Mårdh PA, Odham G, Westerdahl G. Use of selected ion monitoring for detection of tuberculostearic and C32-mycocerosic acids in mycobacteria and in fiveday-old cultures of sputum specimens from patients with pulmonary tuberculosis. Acta Path Microbiol Scand 1981; 89B: 245-51.

Lund, Sweden

PER-ANDERS MARDH LENNART LARSSON

Department of Infectious Diseases and Department of Clinical Microbiology, State Serum Institute,

University Hospital, Copenhagen, Denmark

NIELS HØBY

Tuberculosis Department, State Serum Institute, Copenhagen

HANS CHR. ENGBAEK

Department of Ecological University of Lund

GÖRAN ODHAM

Chemistry,

LABELLED ANTIBODIES FOR TUMOUR LOCALISATION

mycobacteria. 1 ml of CSF was autoclaved, lyophilised, and heated in 1 molll methanolic HCI overnight at 80°C. After extraction by n-hexane, the sample was subjected to thin-layer chromatography. 1 The methyl ester fraction was taken up in ethyl acetate, evaporated to dryness, and diluted to 20 0 with n-hexane, of which 0 - 3 3 pl was used for GC/MS analysis. Splitless injections were made onto a 25 m fused silica capillary column (0 - 2 mm internal diameter), installed in a Carlo Erba gas chromatograph (model 4160)/Ribermag R 10-10 mass spectrometer. The carrier gas through the column was helium, at a flow rate of 1 ml/min. SE-54 was used as stationary phase and the column temperature was programmed up to a final temperature at 200°C. Selected ion monitoring of methyl tuberculostearate was done by chemical iomsation, with ammonia as reactant gas at 1 mm Hg focusing at m/z 330. The temperature of the ion source was 250°C.

Microbiology,

University of Lund,

S:R,—Dr Rainsbury and Dr Westwood (Dec. 11, p. 1347) discuss use of "’In-chelate-labelled antibodies for the radioimmunolocalisation of tumours. While agreeing that i iIn is superior to 131I for this purpose, we disagree with their interpretations elsewhere. Rainsbury and Westwood suggest that iodination impairs the biological activity of antibodies whereas chelate attachment does not, and in support of this they cite their own results. Yet this depends on the methods used and the numbers of iodine or chelate groups inserted into the antibody, information not given in their letter. Iodination causes little or no damage to IgG antibodies4if careful attention is paid to the amounts of oxidising agent and iodide

the

added and to the reaction time. We routinely use chloramine-T to iodinate antibodies and have detected no degrading effect by sensitive radioimmunoassay. In contrast, Krejcarek and Tucker5 showed that chelate labelled albumin was cleared more rapidly from the circulation of mice than was i2sl-albumin. Evidence is limited, but others have confirmed this6,7 with different preparations; Layne et a1.7 showed that incorporation of an average ofO’ 7 DTPA molecules per molecule of fibrinogen resulted in more rapid blood clearance than that of 12SI-fibrinogen. These results suggest that chelate attachment induces greater change than does iodination for similar substitution rates. L, Mårdh PA, Odham G, Westerdahl G. Detection of tuberculostearic acid in biological specimens by means of glass capillary gas chromatography/electron and chemical ionization mass spectrometry, utilizing selected ion monitoring. J Chromatogr Biomed Appl 1980; 182: 402-08. 4. Hunter WM. Radioimmunoassay. In: Weir DM, ed. Handbook of experimental immunology: Vol I, immunochemistry. Oxford: Blackwell 1978: 14.3-14.8. 5. Krejcarek GE, Tucker KL. Covalent attachment of chelating groups to macromolecules. Biochem Biophys Res Comm 1977; 77: 581-85. 6. Leung CSH, Meares CF, Goodwin DA. The attachment of metal-chelating groups to proteins: Tagging of albumin by diazonium coupling and use of the product as radiopharmaceuticals. Int J Appl Radiat Isot 1978; 29: 687-92. 7. Layne WW, Hnatouich DJ, Doherty PW, Childs RL, Lanteigne D, Ansell J. Evaluation of the viability of In-11-labelled DTPA coupled to fibrinogen. J Nucl 3. Larsson

Med 1982; 23: 627-30.

,

368

Rainsbury and Westwood state that because the chelate technique caused less damage to the antibody its performance was enhanced. This is highly unlikely. We think that there are two reasons why 111In chelates may be preferable. Firstly,"In has two gamma emissions (171 and 247 keV) which are more suitable for detection than, the single 364 keV emission of 131 I. Calculation based on the efficiency of conventional LFOV gamma cameras8 indicate a potential eight-fold higher count rate for III In than for 1311. Secondly, indium is stable within cells,9 unlike iodineio which is probably rapidly stripped from protein and diffuses out. Finally, monoclonal antibodies may behave idiosyncratically with respect to the damaging effects of labelling procedures. This should be borne in mind when results with different antibody systems are compared. Immuno Diagnostic Research Laboratory,

Department of Immunology,

D. S. FAIRWEATHER A. R. BRADWELL P. W. DYKES

Medical School,

University of Birmingham, Birmingham B 15 2TJ

DH. Gamma-ray detection efficiency and image resolution in sodium iodide. Rev Sci Ins 1964; 35: 693-97. 9. Thakur ML, Segal AW, Louis L, Welch MJ, Hopkins J, Peters J. Indium-111-labelled cellular blood components: Mechanism of labelling and intracellular location in human neutrophils. J Nucl Med 1977; 18: 1020-24. 10. Stern P, Hagan P, Halpern S, Chen A, et al. The effect of the radiolabel on the kinetics of monoclonal anti-CEA in a nude mouse-human colon tumour model In: Mitchell MS, Oettgen HF, eds Hybridomas in cancer diagnosis and treatment. New York Raven Press, 1982; 245-53. 8.

Anger HO, Davis

Commentary from Westminster Prescribing and Promotional Costs THE medical profession often feels irritated by the media’s capricious attitude to medical issues. One day all the national papers and television get excited about transplants, cancer cures, or drug prices, only to lose interest a few days later. Often the fuss does more harm than good, as it did to the supply of donor organs. But there can be little doubt that newspapers and television have played a key role in forcing the Government to publish the Greenfield reportl on effective prescribing, which examines ways in which the N.H.S. could reduce its drugs bill. In 1980-81 general practitioners prescribed f866 million worth of drugs, chemists were paid 234 million in fees and allowances, and N.H.S. hospitals spent 185 million on drugs. The Social Services Secretary, Mr Norman Fowler, received the report from one of his principal medical officers, Dr Peter Greenfield, exactly a year ago. It lay for ten months gathering dust in Mr Fowler’s office, and the D.H.S.S. plainly had no intention of acting on it. It became known that the report commended the idea of substitution by pharmacists of the generic equivalent of branded drugs prescribed by doctors, but there was still little pressure on the Government to publish the report. About two months ago the Effective

concerted interest in the company profits, and the cost to the N.H.S. This, combined with Opposition pressure in Parliament, finally forced Mr Fowler’s hand. The report is something of an anticlimax, since it sidesteps the most important implications of generic substitution. "Any significant reduction in the prescribing of branded drugs national press started

to

subject of drug damage

take

to

a

patients, drug

1. Report to the Secretary of State for Social Services of the Informal Working Group on Effective Prescribing. Department of Health and Social Security. Copies of the report are available from Mr David Caygill, Department of Health and Social Security, Room 618, Eileen House, 80/94 Newington

Causeway, London SE1

6EF.

could have an adverse effect on the innovative sector of the industry and so limit the money available for research and development," but, it adds, this is a matter for someone else to deal with. Opposition politicians of the Labour Party and the Social Democrats are clear in their belief that generic substitution is something the drug companies will just have to live with. But the Government takes a different attitude. Mr Fowler, without saying so in plain words, is determined not to go down that road. Cabinet colleagues and Departmental advisers, as well as drug industry spokesmen, have pointed out to the Secretary of State that the drug industry employs 70 000 people in Britain and exports C600 million worth of pharmaceuticals each year. Nor should it be forgotten that several drug companies make generous contributions to Tory Party funds (Glaxo gave £ 25 000 last year, and Beecham gave 20 000, to name but two). Mr Fowler has no wish to become the scourge of the drug companies, but at the same time he does not want to be seen to be feather-bedding them, or allowing them to make unreasonable profits out of the N.H.S. His strategy therefore is to bypass the generic substitution issue and turn attention to the Pharmaceutical Price Review Scheme. He prepared the ground a couple of weeks ago, when his Under-Secretary, Mr Geoffrey Finsberg, announced that the Government is to review the way the PPRS works. The objective, Mr Finsberg told Parliament, was to ensure a good balance between the interests of the taxpayer, the patient, and the drug producer. It sounds impressive, but in fact the D.H.S.S. is apparently already well satisfied with the present working of the scheme, as became clear when the Comptroller and Auditor General, Mr Gordon Downey, examined its workings on behalf of the backbench Public Accounts Committee (which will give its own opinions on the PPRS at the beginning of April}. The D.H.S.S. requires drug firms to submit audited annual financial returns which break down sales, costs, profits, and capital employed. If the Department thinks the profits excessive it can direct the firm to cut prices accordingly. Profits are supposed to stay within targets set by the D.H.S.S. The Department told Mr Downey that "on the basis of the criteria agreed with the Treasury for the PPRS the pharmaceutical industry did not earn excessive profits". The PPRS itself "represented good value for money, furnished good quality and safe medicines at reasonable prices, fostered a healthy domestic industry, and contributed to a substantial export surplus", Mr Downey was told. Such faith in the scheme leaves little room for Mr Fowler to make any impression on the N.H.S. drugs bill. But he will have to make a gesture of some sort, since the Public Accounts Committee’s report on the scheme is not expected to be particularly anodyne. When the committee heard from a D.H.S.S. assistant secretary, Mr John Long, that the Department employs only one accountant to check the drug companies’ returns (though he has access to some help) the committee’s alert chairman, Mr Joel Barnett, asked ifMr Long was satisfied that the D.H.S.S. was not sometimes "the victim of creative accounting"? Mr Long admitted he was not satisfied. The committee will undoubtedly call for a more rigorous approach, and could well have some harsh words to say about the drug firms. Mr Fowler may announce, as his response both to Greenfield and to any criticisms from the Public Accounts Committee, that the D.H.S.S. is to strengthen its accounting team. He may well add that drug companies are to be allowed to include less of their promotional costs in the sums allowed