NAFAMOSTAT TO STABILISE PLASMA SAMPLES TAKEN FOR COMPLEMENT MEASUREMENTS

896

fibrinolysis can lead to thrombosis.’ In most healthy people desmopressin stimulates both fibrinolysis and coagulation, increasing plasma levels of tissue plasminogen activator (t-PA) and plasmin activity. However, in the presence of high levels of t-PA inhibition desmopressin infusion increases neither t-PA activity nor plasmin activity.2 Plasma levels of t-PA inhibition are increased in patients with coronary artery disease,3and are predictive of recurrent myocardial infarction.4 It seems reasonable to avoid desmopressin in patients with coronary artery disease. One possible mechanism for the increased risk of thrombosis in such patients could be high levels of t-PA inhibition, which prevent the normal fibrinolytic response to desmopressin. However, the normal t-PA activity response in the Dutch physician with coronary thrombosis suggests that other mechanisms also contribute. Haemophilia and Thrombosis Unit, University Department of Medicine, Royal Infirmary, Glasgow G4 0SF

G. D. O. LOWE

1. Astrup T. Fibrinolysis m the organism. Blood 1956; 11: 781-806. 2. Lowe GDO, Douglas JT, Small M, Forbes CD, Kluft C. Evidence for plasminmediated fibrinolysis after release of tissue-type plasminogen activator by desmopressin infusion. In: Lowe GDO, Douglas JT, Forbes CD, Henschen A, eds. Fibrinogen 2: biochemistry, physiology and clinical relevance. Amsterdam: Excerpta Medica, 1987: 285-88. 3. Paramo JA, Colucci M, Collen D, van de Werf F. Plasminogen activator inhibitor in the blood of patients with coronary artery disease. Br Med J 1985; 291: 573. 4. Hamsten A, Wiman B, DeFaire U, et al. Increased plasma levels of a rapid inhibitor of tissue plasminogen activator in young survivors of myocardial infarction. N Engl J Med 1985; 313: 1557-63.

ANTACIDS AND DRUG TRIALS FOR DUODENAL ULCER

SIR,-Professor Lam (Feb 18, p 384) comments that the 59% duodenal ulcer healing rate after 8 weeks on treatment with cimetidine 400 mg twice daily cited in Dr Marshall and his colleagues’ paper (Dec 24/31, p 1438) is incorrect and should be about 90%. Clinical trials of Hz-blockers for duodenal ulcer healing have allowed the use of antacid tablets as required. Marshall’s patients "were cautioned not to take antacids". The interaction between small doses of antacids and Hz-blockers on neutralisation of gastric acid, protection of duodenal mucosa, and impact on duodenal ulcer healing is not known. In in-vitro experiments Lam et all found that two antacid tablets (800 mg) neutralised 25 mmol hydrochloric acid (0’11 mmol/1) in 150 min, a neutralising capacity close to that of a similar antacid liquid preparation. Peterson and co-workersz reported that cimetidine 300 mg four times a day plus antacid seven times a day was significantly better than cimetidine alone in raising gastric pH. Reduction of gastric acidity was similarly obtained with cimetidine 600 mg twice daily plus antacids four times a day, and this combination was very effective in reducing nocturnal gastric acidity.3 In a double-blind, randomised, multicentre trial 4 weeks of treatment with low doses of antacids (one tablet four times daily) resulted in a duodenal ulcer healing rate of 71-1%, similar to the 78 -4 % obtained with 800 mg cimetidine.4 When evaluating ulcer healing rates in trials with Hz-blockers in a heterogeneous group such as duodenal ulcer patients, the confounding variable of antacid tablets should be allowed for. Only then will we know whether healing rates lie close to 59% or to 90%. Division of Gastrointestinal and Coagulation Drug Products, Center for Drug Evaluation and Research, Food and Drug Administration, Rockville, Maryland 20857, USA

NAFAMOSTAT TO STABILISE PLASMA SAMPLES TAKEN FOR COMPLEMENT MEASUREMENTS

SIR,-Since its discovery by Bordet in 1899, as a system of cooperative serum factors necessary for the lysis of cells by antibody, our knowledge of complement and complement activation has grown at an enormous pace. Excessive activation of the various complement pathways is almost inevitably harmful (eg, in rheumatoid diseases, renal disease, cirrhosis, and septicaemia). One current focus is the measurement of the biologically active anaphylatoxins C3a, C4a, and C5a; several excellent radioimmunoassay kits are available. Unfortunately their value is considerably decreased by in-vitro activation of plasma samples even when these are collected into edetic acid (EDTA) and separated and stored under ideal conditions. The immediate assay of fresh samples poses logistical problems and is prohibitively expensive. We have been evaluating the addition of a synthetic protease inhibitor, nafamostat mesylate (’Futhan’; Amersham International) which, when added to EDTA in the blood sampling tube, greatly increases the stability of complement components. Blood samples (20 ml) were obtained from volunteers and from patients, who gave informed consent. The samples were immediately split into tubes containing EDTA with or without nafamostat and plasma was separated and stored at -20°C. The assays were done in parallel within 2 weeks of collection, using Amersham kits. In controls (50 volunteers from Amersham and from the Royal Hallamshire Hospital) there was no measurable C5a and narrow ranges for both C3a and C4a levels.1 95th percentile ranges in the nafamostat-treated samples were C3a 102-212 ng/ml and C4a 0-571 ng/ml, roughly half those recorded in plasma with EDTA alone (C3a 77-491 ng/1, C4a 0-1683 ng/1). Activities in plasma with EDTA alone depended on the number of freeze-thaw cycles, doubling for each cycle, nafamostat samples were highly stable to such abuse. Clinical studies were done on groups of 20-30 patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), renal disease, liver disease, and a variety of vasculitic conditions including scleroderma. The patients with SLE and RA were thought to be clinically controlled and we present here the data

R. PRIZONT S. B. FREDD

SK, Lam KC, Lai CL, et al. Treatment of duodenal ulcer with antacids and sulfipiride. Gastroenterology 1979; 76: 315-22. 2. Peterson WL, Barnett C, Feldman M Reduction of twenty-four hour gastric acidity with combination drug therapy in patients with duodenal ulcer. Gastroenterology 1979, 77: 1015-20. 3. Mahachai V, Jamali F, Thomson ABR. Effect of combination of antacid and cimetidine on 24-hour intragastric acidity in patients with asymptomatic duodenal ulcer. Clin Ther 1984; 6: 808-23 4. Weberg R, Aubert E, Dahlberg O, et al. Low dose antacids or cimetidine for duodenal ulcer? Gastroenterology 1988; 95: 1465-69. 1. Lam

Plasma C3a anaphylatoxin levels in health and disease.

0 EDTA alone; . nafamostat/EDTA. (Several EDTA samples gave readings in excess of 1000 ng/ml: the computer records all such data as 1000 and a single point at this level may represent several patients.) =

=

897

anaphylatoxin levels in these two groups and in controls (figure). Similar results were obtained for C4a, while C5a was not quantifiable (below 10 ng/ml). The figure shows that while anaphylatoxins measured in EDTA plasma give some indication of disease activity, this is badly impeded by what must be further in-

for C3a

vitro activation. However, the

narrow

normal range in the

inhibitor-treated plasmas (figure) strongly suggests that many of the

patients have active disease despite no clinical signs. These are preliminary results, requiring further study and follow-up assays on selected patients. Nevertheless, the differences in results between EDTA/nafamostat samples and those stabilised with EDTA alone are startling. This inhibitor may enable us to examine early disease activity in a variety of conditions. Furthermore, samples can now be transported to distant laboratories with specialised assay facilities with little fear of in-vitro artifact. Clinicians wishing

to

evaluate nafamostat mesylate should

Department of Immunology, Royal Hallamshire Hospital, Sheffield S10 2JF Amersham International plc, Aylesbury, Bucks HP20 2PT

contact

S. S.

JOHN WATKINS

deformability of the cells. Thus treatment of normal erythrocytes with the permeant sulphydryl reagent N-ethylmaleimide results in an increase in the ratio of SpD:SpT to a degree similar to that observed in HPP cells and a corresponding partial resistance to invasion by P falciparum in vitro, which paralleled the increase in SpD,5 although these results contrast with those reported in another study.7 The West African variant cells described by Dhermy et al might, like the Spcx1/74 variants, be partly resistant to invasion. The Nigerian patient with "ovalocytosis" and intense P falciparum malaria described by Dr Fleming (Oct 8) may well be an HE variant with normal spectrin tetramer formation,3 which we have shown to be fully susceptible to invasion.5 Additionally some HE and HPP symptom-free carriers do not show any erythrocyte morphological alteration on blood filins and would consequently only be detected following molecular analysis of the red cell membrane. Department of Haematology, London Hospital Medical College, London El 2AD

CHRISTINE A. FACER

GRAEME WILD STEVEN SMITH

MALARIA, HEREDITARY ELLIPTOCYTOSIS, AND PYROPOIKILOCYTOSIS

SIR,-I read with interest recent Lancet correspondence on malaria and hereditary elliptocytosis in Africa (Oct 8, p 857; Jan 28, p 225). There are two points I would like to raise. Hereditary elliptocytosis (HE) is a heterogeneous group of disorders in terms of inheritance, red cell morphology, severity of haemolysis, and underlying molecular defect.The variants include

asymptomatic or symptomatic HE, hereditary pyropoikilocytosis (HPP), spherocytic HE, and the stomatocytic elliptocytosis or Melanesian ovalocytosis referred to in your Sept 10 editorial. Melanesian ovalocytosis can be distinguished morphologically from other forms of elliptocytes because the elliptocytes are usually spoon-shaped and their folding in blood smears produces a transverse stoma (hence the designation "stomatocytic" HE).2 The molecular defect responsible for the increased rigidity of Melanesian ovalocytes is unknown. The clinical heterogeneity of HE/HPP syndrome is paralleled by a difference in the molecular defect within the membrane of these cells. The most common defect, found in about 30% of HE patients and all HPP patients in an abnormal spectrin dimer (SpD) self-association in which the abnormality is localised on the (’,(1 domain of spectrin representing the SpD-SpD contact site.3 Several spectrin variants have been described’ which result in defective SpD-SpD association into tetramers, producing an unstable and less defonnable cell. The amount of mutant spectrin in the cell and the severity of the spectrin self-association dictates the seriousness of HE and HPP. The spectrin variants SPo/65 and Sp(’,(1/46 in West African populations described by Dr Dhermy and his colleagues (Jan 28), may be relevant to partial protection against malaria. For example, in collaboration with Dr J. Palek and Dr J. Prchal (St Elizabeth’s Hospital of Boston) we have tested erythrocytes from nine patients from two unrelated black families with HPP or the HPP asymptomatic carrier state for susceptibility to invasion by Plasmodium falciparum merozoites (Wellcome/Liverpool West African strain) in vitro.s The molecular defect had previously been characterised as an Spxl/74 mutant with a variable degree of spectrin dimerisation .6 Although resistance to invasion was never absolute invasion was, in all but one sample, significantly reduced, varying from 38 %-71 % of the invasion observed into normal cells. Red cells taken from two donors, sampled and tested on different occasions, gave remarkably repeatable results.4 One explanation for the reduced invasion seen in HPP cells relates to the increased SpD within the membrane of these cells (rather than the 30% decrease in spectrin content, also a feature of HPP) and the resulting decreased

1. Coetzer T, Lawler J, Prchal JT, Palek J. Molecular determinants of clinical expression of hereditary elliptocytosis and pyropoikilocytosis. Blood 1987; 70: 766-72. 2. Lux SE. Disorders of the membrane skeleton: hereditary spherocytosis and hereditary elliptocytosis. In: Stanbury JB, et al, eds. The metabolic basis of inherited diseases. New York: McGraw-Hill, 1983: 1573. 3. Palek J. Hereditary elliptocytosis and related disorders. Clin Haematol 1985; 14: 45-87. 4. Palek J. Hereditary elliptocytosis, spherocytosis and related disorders: consequences of a deficiency or a mutation of the membrane skeletal protein. Blood Rev 1987; 1: 147-68. 5. Facer CA. The red cell cytoskeleton and invasion by malana parasites. Mem Inst Oswaldo Cruz 1986; 81: 111-14. 6. Liu S-C, Palek J, Prchal J, Castleberry RP. Altered spectrin dimer-dimer association and instability of erythrocyte membrane skeletons in hereditary pyropoikilocytosis. J Clin Invest 1981; 68: 597-605. 7. Rangachari K, Beaven GH, Clough B, et al. A study of red cell membrane properties in relation to malarial invasion. Mol Biochem Parasitol (in press).

PRAZIQUANTEL FOR NEUROCYSTICERCOSIS SIR,-Dr Moodley and Professor Moosa (Feb 4, p 262) question whether praziquantel is really "the new hope" for patients with neurocysticercosis. They contrast our studies1,2 and those of other groups3 reporting success with this drug in parenchymal neurocysticercosis, with the experience of themselves and others of spontaneous resolution of neurocysticercosis lesions in children.’ we have reported, some of these lesions do resolve spontaneously, especially when the cyst is surrounded by signs of inflamITlation.1,7 However, neurocysticercosis is rare in children and very frequent in adults and it is different in the two age groups.b In children it is often benign and infection with cysticerci is usually followed by an intense immunological response that imposes limits on the disease, in most cases only one cysticercus is found in brain parenchyma, surrounded by inflammation and oedema, and the only clinical sign is epilepsy, and with this picture the disease is self-limiting.7 However, in adults neurocysticercosis is far more frequent and usually runs a chronic and severe courseand praziquantehz (and albendazole8) have greatly improved the prognosis.9

As

National Institute of Neurology and Neurosurgery, 14410 Mexico 22, DF

JULIO SOTELO

F, Rodriguez J, Torres B, Rubio F. Therapy of parenchymal brain praziquantel N Engl J Med 1984; 310: 1001-07. 2 Sotelo J, Escobedo F, Penagos P Albendazole vs praziquantel for therapy of neurocysticercosis, a controlled trial. Arch Neurol 1988; 45: 532-34 3. Van Dellen JR, McKeown CP Praziquantel in active cerebral cysticercosis. J Neurosurg 1988; 22: 92-96 4. Dawood A, Moosa A. Cerebral cysticercosis in children. J Trop Pediatr 1984; 30: 1 Sotelo J, Escobedo

cysticercosis with

136-39. 5. Thomson 6.

7

8. 9.

AJ, De Villers JC, Moosa A, Van Dellen JR. Cerebral cysticercosis in children in South Afnca Ann Trop Pediatr 1984; 4: 67-77. Mitchell WG, Crawford TO Intraparenchymal cerebral cysticercosis in children, diagnosis and treatment Pediatrics 1988; 82: 76-82 Sotelo J, Guerrero V, Rubio F Neurocysticercosis: a new classification based on active and inactive forms. a study of 753 cases. Arch Intern Med 1985; 145: 442-45. Sotelo J, Penagos P, Escobedo F, Del Brutto OH. Short course of albendazole therapy for neurocysticercosis. Arch Neurol 1988; 45: 1130-33. Del Brutto OH, Sotelo J Neurocysticercosis. an update Rev Infect Dis 1988; 10: 1075-87