684 LENGTH OF DISTAL VALVULAR CATHETERS IN HYDROCEPHALIC CHILDREN
SIR,-In hydrocephalic children given a shunt, the growth of the patient may displace the inferior end of the distal valvu lar catheter from the cardiac or peritoneal cavity and stop it functioning.1-5 To find out how much extra tubing should be left in place to avoid this complication we measured the correlation between the distance from the mastoidal region to the xiphoid appendix (the approximate distance the distal valvular catheter must cover) and height in 200 girls and boys (from newborns to 13-year olds). The correlation coefficient was 0.976 (95% confidence limits 0--967-0-982) and the regression line, which had a slope of 0.18 (0175-0185), was defined by the equation Y=0.18X+4.02 where Y was the distance the distal catheter had to cover in cm and X was the child’s height in cm. Thus, the length of catheter which should be placed in a child to account for his growth could be calculated from the equation, assigning X the child’s height when full-grown. Full-grown height can be predicted from parental height.6 It is impossible to leave the excess length of catheter inside the cardiac cavity, but there may be room for it in the peritoneal cavity without causing complications. Department of Pædiatrics, University of Santiago de Compostela, Hospital General de Galicia, Santiago de Compostela, Spain
M. CASTRO GAGO R. TOJO
J. M. PAZ M. POMBO
MARROW BUFFY COAT AND BIOPSY
SIR,—Percutaneous trephine biopsy, using either the Westerman-Jensen or the Jamshidi needle, is now a well-established technique for demonstrating infiltrative lesions in the bone-marrow. In a personal series now exceeding 5000 combined aspiration and trephine biopsies we have only twice identified lesions in aspirated material not present in an adequate trephine biopsy and we would therefore support the view7 that biopsy remains the technique of choice for detecting metastatic disease. However, we have developed a modification applicable to the preparation of marrow buffy coat which offers two advantages over the trephine: cytological detail is better preserved and cytochemical studies may conveniently be carried out on larger volumes of tissue than are readily available for the buffy-coat preparation. Trephine is done from the posterior superior iliac spine and a minimum of 3 cm core of tissue obtained, if necessary, by repeat biopsy through the same skin wound but with the needle inclined on one occasion superiorly and on the next inferiorly. 5 ml of marrow-rich blood is collected with strong aspiration into a dry syringe and immediately delivered into a plastic tube 1 x 10 cm. Before the blood has had time to clot, the tube is centrifuged for 10 min at 200 g during which the marrow particles rise to the top and are firmly trapped in the fibrin mesh. This upper layer is freed from the other material in the tube and provides a dense collection of particles suitable for cytochemical or ultrastructural studies which is available for rapid processing and histological examination since there is no need for decalcification. It was in this type of preparation that a solitary granuloma was observed in one patient and myeloma demonstrated in a second when, on each occasion, the trephine biopsy was negative. These preparations are free of red cells but when particles 1. 2.
3. 4. 5. 6. 7.
Berney, J. Med. Hyg. 1973, 1074, 1540. Eckstein, H. B., Macnab, G. H. Lancet, 1966, i, 842. Illingworth, R. D., Logue, V., Symon, L., Hemura, K. J. Neurosurg. 1971, 5, 681. Isamat, F. Rev. Esp. Pediat. 1971, 158, 196. Pertnisef, B., Bounal, J., Cabezas, C. Neurochirurgie, 1964, 10, 43. Roche, A., Wainer, H., Thissen, D. Pediatrics, 1975, 56, 1026. Ellman, L. Am. J. Med. 1977, 62, 163.
are scanty an artefact may be produced in which lymphocytes and granulocytes form separate layers. However, to those familiar with this pattern this poses no diagnostic difficulties.
University of Cape Town and Groote Schuur Hospital, Observatory, Cape Town, South Africa
PETER JACOBS LEONARD B. KAHN
SPECIFIC DETECTION OF IgM-ANTIBODIES BY ELISA, APPLIED IN HEPATITIS-A
SIR,-In the early diagnosis of infectious diseases the detection of specific IgM antibodies is becoming more important. This is usually done by separating IgM from IgG by the cumbersome sucrose-density gradient centrifugation or by absorption of IgG and/or the use of an anti-IgM conjugate. The use of such a conjugate may give rise to false-positive reactions due 2 to rheumatoid factor. 1 We have developed a sensitive, rapid, and simple microscale enzyme-linked immunosorbent assay (ELISA) for the detection of specific anti-hepatitis-A IgM antibodies (anti-HAV-IgM) based on the use of anti-human-IgM (µ chain) for coating microtitre plates. Interference with rheumatoid factor is reduced by using an anti-human IgM for coating microtitre plates (in ELISA) and anti-antigen antibodies for the preparation of con-
jugate. Rabbit anti-human-IgM serum (no. 10-091, Dako, Copenhagen), diluted 1:500 in 0-01 mol/l phosphate-buffered saline (PH 7-2) or a sheep anti-human-IgM-:;erum diluted 1:5000 was used to coat the wells of polyvinylchloride or polystvrene microtitre plates. Then the IgM present in the test serum was allowed to react. Sera were diluted, ranging from 1: 102 to 1:10’.Thereafter a fixed amount of hepatitis A virus (HAV) was added, in the form of a 0.25% extract of a strongly positive fæcal sample (giving an extinction of 1.100 in the sandwich HA’ test. HAV will bind to the specific anti-HAV-IgM, and horseradishperoxidase-coupled anti-HAV ;(conjugate) is then allowed to react with the bound HAB’. A mixture of o-phenylenediamine and urea peroxide is used as enzyme substrate, giving a colour reaction measurable in a photometer at 492 nm. Samples with extinctions greater than or equal to the mean extinction of five negative controls+Sxs.D. were regarded as positive. The titre was defined as the maximum dilution giving a positive reaction. Titres up to 1 :4.4x 106 for strongly positive sera were found. Ten coded serum samples, kindly provided by Mr R. v. d. Akker (Laboratory of Virology, Dutch Natioral Institute of Health, Bilthoven) were tested in a 1:100 dilution in our ELISA. Results were compared with those obtained in the sandwich inhibition ELISA for the detection of anti-HAV3 on IgM-fractions prepared by sucrose-density gradient centrifugation. Two sera were strongly positive for rheumatoid factor with titres 1:1024 (Rose-Waaler test), and one serum had an anti-HAV-IgG titre of 1: 105.
The results (see table) show a high specificity for the detection of anti-HAV-IgM. Neither rheumatoid factor, nor hightitre anti-HAV-IgG gave rise to false-positive reactions. The figure illustrates the course of hepatitis A in one patient. Fxcal samples were tested for HAV in the sandwich ELISAand sequential sera were tested for anti-HAV in the sandwich-inhibition ELISAand for anti-HAV-IgM. The patient received 0.7 ml human-immune-serum globulin (0-02 ml/kg body-weight) one week after the assumed day of infection. Seroconversion was then seen in the anti-HAV test with a titre of 1:40. Anti-HAV-IgM was negative. Three days before the onset of illness the anti-HAV titre was 1:2000 and the antiHAV-IgM titre 1:3000. The anti-HAV-IgM titre rose to 1 :4 x 104 (estimated) at the first day of illness to a maximum of 1:4 4 x 104 two weeks later. HAV was excreted from 2 to 5 weeks after infection, and hepatitis developed 4 weeks postinfection. The anti-HAV-IgM ELISA is a highly sensitive test that will find its application in rapid diagnosis of hepatitis A. The principle of the test may also be applicable to detection of specific 1.
Gispen, R, Nagel, J., Brand-Saathof, B., de Graaf, S. Clin. exp. Immun. 1975, 22, 431. 2. Yeni, P., Segond, P, Massias, P., Pillot, J. Lancet, 1978, i, 219. 3. Duermeyer, W., v. d. Veen, J., Koster, B. ibid. 1978, i, 823.
685 COMPARATIVE STUDY OF
ACTIVATION OF RHEUMATOID FACTOR DURING PREGNANCY
SIR,—The IgM-class rheumatoid factor (R.F.) can cause false-positive results when IgM antibodies in human serum are detected by labelled anti-human-IgM immunoglobulins in, for instance, immunofluorescence or radioimmunoassay (R.I.A.) tests.’2 This is a serious problem, especially when pregnant women are
examined for can lead
rubella infection, since a falseunnecessary abortion or at least
the patient anxiety. We have lately seen few such false
positives result in our for rubella IgM antibodies,2 but these patients have almost invariably been pregnant. Since the prevalence of high R.F. titres in a normal population was low3 and since acute rubella infection did not activate R.F. in males,4 we investigated the effect of pregnancy on R.F. production. The R.F. levels of 88 pregnant women (mean age 27, range 17-37) were determined by a solid-phase R.I.A.3 and compared with R.F. levels of 111 female blood-donors (see figure). The median value for blood-donors was 5 R.F. units and for pregKendall’s S test). Included in nant women 35 units (P