Diagnostic difficulties of HCV serological tests

116

[SD 4-6] years, range 28-44; number of previously attempts 2-5 [1-5], 1--6; number of previously transferred embryos 7-2 ,1-19), which amounts to a clinical pregnancy rate of 67% and an implantation rate of 29%. Tadir and colleagues’ pioneering work was based on experimental research, whereas the erbium-YAG laser has already repeatedly proved its qualities in clinical appliance.

mean

age 35-5

failed IVF

Institute of Sterility Treatment, A-1130 Vienna, Austria

WILFRIED FEICHTINGER HEINZ STROHMER KARL M. RADNER

Kaufmann R, Hibst R. Pulsed Er:YAG- and 308 nm UV-excimer laser: an in vitro and in vivo study of skin-ablative effects. Lasers Surg Med 1989; 9: 132-40. 2. Rasmussen RE, Hammer-Wilson M, Berns MW. Mutation and sister chromatid exchange induction in Chinese hamster ovary (CHO) cells by pulsed excimer laser 1.

radiation at 193 nm and 308 nm and continuous UV radiation at 254 nm. Photochem Photobiol 1989; 49: 413-18. 3. Strohmer H, Feichtinger W. Successful clinical application of laser for micromanipulation in an in vitro fertilization program. Fertil Steril (in press).

Cryopreservation of embryos and

pregnancy

rates after IVF

SIR,-Dr Tan and colleagues (June 6, p 1390) demonstrate that the cumulative pregnancy rate after repeated IVF treatment declines after the female partner reaches the age of 34 years. The cryopreservation of embryos after IVF is a safe and cost-effective 1 way to increase cumulative pregnancy rates per oocyte recovery, do not include this in their results. yet they PREGNANCY RATES PER TRANSFER OF CRYOPRESERVED EMBRYOS IN NATURAL CYCLES (NC) OR CYCLES WITH HORMONE REPLACEMENT THERAPY (HRT).

colleagues in the University of Kansas School of Medicine in the 1920s, and they showed that a bolus injection of 30-50 mg/kg methylguanidine sulphate resulted in a large (greater than 100 mm Hg) sustained rise in blood pressure, similar to that seen with inhibitors of NO synthase.4 The results were consistent in over 200 experiments. These experiments were done to explain why patients with renal disease developed hypertension, the theory being that a pressor product of metabolism may be retained in these patients. Such studies influenced Goldblatt and colleagues,’ who believed that retention of guanidine compounds was one explanation for their observation that renal ischaemia causes hypertension. Since the dose Taylor and colleagues used was only slightly lower than that used by Major it seems that there may be inter-species differences in sensitivity of the endothelial constitutive NO synthase to this substance. Furthermore, the demonstration that aminoguanidine is an effective inhibitor of the inducible NO synthase6 invalidates the presumption that the full aminoacid structure is needed to inhibit NO synthase. Despite the thoughts of Taylor and colleagues, it seems that small molecules that resemble the guanidino end of the arginine molecule may in fact be important modulators of NO synthesis and deserve further study. Departement of Medicine and Therapeutics, University of Aberdeen,

NIGEL BENJAMIN

Foresterhill, Aberdeen AB9 2ZD, UK

Major RH. Relationship between certain products of metabolism and arterial hypertension. JAMA 1924; 83: 81-84. 2. Major RH. The possible relationship between guanidine and high blood pressure. Am J Med Sci 1925; 170: 228-32. 3. Major RH, Weber CJ. The effect of methylguanidine upon the blood pressure of adrenalectomized dogs. J Pharmacol Exp Ther 1929; 35: 351-54. 4. Aisaka K, Gross SS, Steinberg C, Griffith OW, Levi R. NG methylarginine, an inhibitor of endothelium-derived nitric oxide synthesis, is a potent pressor agent m the guinea pig: does nitric oxide regulate blood pressure in vivo? Biochem Biophys 1.

Res Comm 1989; 160: 881 5. Goldblatt H, Lynch J, Hanzal RF, Summerville WW. The production of persistent elevation of systolic blood pressure by means of renal ischaemia. J Exp Med 1934; 59: 347-78. 6. Corbett JA, Tilton RG, Chang K, et al. Aminoguanidine, a novel inhibitor of nitric oxide formation, prevents diabetic vascular dysfunction. Diabetes 1992; 41: 552-56.

*p < 0 05 both

<

34 yr and 35-39 yr groups.

We have found

a

reduced pregnancy

rate

after the transfer of

embryos cryopreserved in propanediol only in women aged 40 years or more (table). This reduction is seen both when embryos are transferred in unstimulated cycles in which replacement is timed relative to the endogenous LH surge, and in cycles in which the endometrium is artificially stimulated by exogenous steroids after pituitary suppression with buserelin therapy, removing the possibility of an impairment of ovarian activity affecting endometrial development. We believe that it is essential to provide cryopreservation facilities in IVF units. Manchester Fertility Services, Manchester BUPA Hospital, Whalley Range, Manchester M168AJ, UK 1.

BRIAN A. LIEBERMAN STEPHEN A. TROUP PHILLIP L. MATSON

Troup SA, Matson PL, Critchlow JD, et al. Cryopreservation of human embryos at the pronucleate, early cleavage or expanded blastocyst stages. Eur J Obstet Gynecol Reprod Biol 1990; 38: 133-39.

Modulators of nitric oxide synthesis SIR,--4Commenting on Dr Vallance and colleagues’ (March 7, 572) discovery of an endogenous inhibitor of nitric oxide (NO) synthesis, Dr Taylor and colleagues (May 16, p 1243) point out the structural similarity of the endogenous metabolite methylguanidine with the guanidino end of the NO synthase inhibitor L-NG monomethyl arginine. They say that there are no reports of methylguanidine having a pressor action. There are in fact many, if somewhat ancient, publications (for example, refs 1-3) on the effects of guanidine compounds, including methylguanidine and dimethylguanidine, on blood pressure in the anaesthetised dog. These experiments were done by Ralph H. Major and his p

Diagnostic difficulties of

HCV serological

tests

SiR,—Dr Allain and colleagues (May 9, p 1171) suggest that "half of all HCV RIBA indeterminate results are likely to be false reactions". Perhaps this is so, depending on the group of patients you choose to examine. A new test (Chiron RIBA HCV SIA prototype) was used to assay 27 samples that had given an indeterminate result by the immunoblot Chiron RIBA HCV test system. This new test uses three recombinant antigens (c33, NS5, and SOD) and two synthetic peptide bands (clOO and c22). According to the manufacturer’s instructions, it should be used only to establish whether indeterminate samples indeed contain specific antibodies to epitopes carried by HCV. The results were: /?/M HCV RIBA C SIA 5M prototype pmfofype PosJindJneg c33 12/1/0 c22 4/7/3 Pos=positme, !nd=!ndetermmate, new= negative

Total 13 14

significant difference between the samples with or c22 (X2 test, p < 0005). reactivity Most patients (10 of 13) included in the c33 group were on haemodialysis and had been transfused several times. It is noteworthy that 7 c22 reactive samples remained indeterminate

There

was

to

a

c33

with the new test, whereas only 1 of c33 reactive samples did so. The c33 band seems to be more specific than the c22 band. c22-only reactivity could be residual,l but in 3 cases it disappeared when the new test was used. Obviously, the difficulty of indeterminate test results persists whichever RIBA is used. The range of such results might increase from 16% with first generation RIBAs2 to 29% with this third generation RIBA. We have to wait for the development of a more

117

satisfactory serological technique since at present PCR technology available in most clinical laboratories. HCV has eluded serological detection for more than a decade and probably will continue to be elusive and to produce conflicting results. is

not

Departments of Microbiology and Paediatrics,

Hospital Ramon Y Cajal, Madrid 28034, Spain

M. MATEOS S. BALLESTERO A. M. POLANCO C. CAMARERO

M, Martos I, Moreno R, et al. Immunoblot assay as diagnostic test for hepatitis C virus infection. J Hepatol 1992; 14: 411-12. 2. Leon A, Canton R, Elia M, et al. Second generation RIBA to confirm diagnosis of HCV infection. Lancet 1991; 337: 912. 1. Mateos

RIBA-2 band intensity and PCR in HCV infection SIR,-Dr Irving and colleagues (June 6, p 1425) show that the absence of c33c or c22-3 band in donors confirmed positive for hepatitis C virus (HCV) antibodies by second generation recombinant immunoblot assay (RIBA-2, Chiron) was significantly more likely to be associated with negative PCR. They suggest that this was due either to a less virulent HCV strain with a different 5’ non-coding region (NCR) or to host factors that help in the elimination of virus replication. They excluded RIBA-2 indetenninate samples from their analysis. A significant proportion of these, in particular those with only c22-3 band, have proved viraemic by PCR.1 We feel that by excluding this group, important information is lost. RELATION BETWEEN RIBA-2 BAND INTENSITY AND PCR FINDINGS

*Number of -

samples

with each band

tFisher’s exact test

+ve=poSltlve,

ve=negaUve.

We have done nested PCR with the same 5’ NCR primers2 on 27 samples, 15 of which were RIBA-2 reactive (two bands or more) and 12 were indeterminate (one band only). The intensity of each band was rated according to the manufacturer’s recommendation from 1 + to 4 + before PCR. The table shows the relation between band intensity and PCR findings. A c22-3 band with intensity of 3+ or above was significantly more likely to be PCR positive. No significant associations were seen with the other bands. Of the 12 indeterminate samples, 8 were c22-3 only. 5 of these were 3 + or above and all were PCR positive, whereas none of the 2 + or less samples were PCR positive. These results suggest that the intensity of the c22-3 band could be used as a guide to viraemic status. We believe that it is still essential to do PCR on all indeterminate cases, but a single and strong c22-3 band should prompt more thorough

investigations and follow-up. that

indeterminate by RIBA-2 but

negative by

PCR could be false-positives in RIBA-2. However,

one cannot

Samples

were

exclude the possibility of virus variants or host factors as Irving and colleagues suggest. On the other hand, samples reactive in RIBA-2 with two or more bands but negative by PCR might be serological

false-positives. Newcastle Public Health Laboratory, Newcastle General Hospital, Newcastle upon Tyne NE4 6BE, UK

C. Y. W. TONG A. A. CODD

Chan S-W, Simmonds P, McOmish F, et al. Serological responses to infection with three different types of hepatitis C virus. Lancet 1991; 338: 1391. 2. Garson JA, Ring CJA, Tuke PW. Improvement of HCV genome detection with "short" PCR products. Lancet 1991; 338: 1466-67. 1

Antiphospholipid antibodies and

recurrent

thrombo-occlusive events SIR,-Dr Sletnes and colleagues (Feb 22, p 451) report a lack of association of IgG or IgM antiphospholipid antibodies (aPL) with increased risk of mortality, reinfarction, or non-haemorrhagic stroke in patients surviving myocardial infarction. However, several methodological concerns should temper their conclusions. The study was not specifically designed to investigate the relation of aPL and recurrent thrombo-occlusive disease. The patients were drawn from the placebo group of a treatment trial to evaluate the efficacy of warfarin after myocardial infarction. Patients were 75 years old or younger, should not have a risk of bleeding, and, most importantly, were excluded if they were thought to require anticoagulation, have atrial fibrillation, valvular heart disease, atrial embolism, and venous thrombosis. Because of this, the patients with true aPL syndrome1 were likely to be excluded from the study. By excluding patients with many features of aPL syndrome, especially other thrombotic events and valvular heart disease, Sletnes et al have, unfortunately, biased their study towards a negative result. In negative studies, it is always important to know the power of the study to detect a clinically important difference. We suspect that this study had only limited power to detect a two-fold increase in stroke risk among the myocardial infarction survivors who had anticardiolipin antibodies. High aPL values, specifically anticardiolipin antibodies, have been shown to be an independent risk factor for first ischaemic stroke in a case-control study.2 They are also associated with a high risk for recurrent cerebral ischaemic events in patients presenting with focal brain ischaemia.3 Perhaps anticephalin antibodies, as a measure of aPL, are not a good marker for recurrent thromboocclusive disease. It is also possible, in the presence of aPL, that there is a greater risk for recurrent ischaemic events after cerebral ischaemia than after myocardial infarction. The jury is still out on aPL as a prognostic marker. Center for Stroke Research, Department of Neurology, Henry Ford Hospital and Health Science Center, Detroit, Michigan 48202, USA

STEVEN R. LEVINE

Department of Medicine (Neurology), University of Texas Health Science Center, San Antonio, Texas

ROBIN L. BREY

Department of Neurology and Epidemiology, University of Maryland, Baltimore, Maryland

STEVEN J. KITTNER

1. Hams EN. Antiphospholipid antibodies. Br J Haematol 1990; 74: 1-9. 2. Antiphospholipid Antibodies in Stroke Study Group (APASS). The association of anticardiolipin antibodies with first ischemic stroke: a multi-center case-control study. Stroke 1992; 23: 161. 3. Levine SR, Brey RL, Joseph CLM, Havstad S. Risk of recurrent thromboembolic events in patients with focal cerebral ischemia and antiphospholipid antibodies. Stroke 1992; 23 (suppl I): 129-132.

** This letter has been shown to Dr Sletnes and colleagues, whose reply follows.-ED. L. SiR,—The objective of our cohort study was to find whether antiphospholipid antibodies (aPL) detected as anticephalin (aCEPHA) and anticardiolipin (aCL) antibodies, were independent risk factors for subsequent mortality, recurrent myocardial infarction, or non-haemorrhagic stroke in an unselected group of patients who had survived an acute myocardial infarction (AMI). The patient cohort studied was the placebo group of the Warfarin Re-Infarction Study. Patients were recruited from five hospitals serving the Oslo area. The decision to register a patient in the study was left to physicians working in these hospitals during the inclusion period. 1918 patients of both sexes who were 75 years of age or younger at the time of discharge from hospital were potentially eligible. Of these, 270 were excluded because they refused to participate. Total mortality in this group was higher than in the placebo group (ie, our study cohort), but the rate of cardiovascular events was lower.2 15 were excluded because of malignant disease, 16 because they died before randomisation, and 65 because they had a permanent residence outside the study area or were physically or mentally unable to comply with the study regimen. 182 were