819 endemic areas and could not have acquired the disease, because transmission of the disease does not occur in the U.K. (the climate is too cold) and because no histological changes associated with filariasis were seen in the lymphatics. We agree, however, that the word "identical" is misleading; what the figure shows is a section through part of a worm in an alveolus, which is certainly a contaminant and not a pathogen. Tubifex and free-living nematodes can contaminate histological sections, and the worm shown in the figure could be a nematode because these worms can sometimes inhabit water taps and can get onto histological sections as contaminants. Department of Medical Microbiology, St. Bartholomew’s Hospital, London EC1 Thames Water, London EC1
SOAD
TABAQCHALI
JANET K. STEVENS
Community Health Services, City and Hackney Health District, London E9
C. GAZIDIS
ELISA FOR ROTAVIRUS
SIR,-Since the publication of ou’ article on an enzymelinked immunosorbent assay (ELISA)1 we have received many requests for anti-rotavirus sera. To generate a large volume of this reagent, we immunised and obtained sera from a goat. While this serum can be conjugated and used as in our original technique better results are obtained with a two-antibody ELISA similar to that used in our detection system for heatlabile Escherichia coli toxin.2 After pretreatment of wells with goat anti-rotavirus antibody diluted 1/100 000 and addition of the stool specimen to be tested, an unlabelled guineapig antirotavirus antibody diluted 1/6000 is added, followed by an alkaline-phosphatase-labelled goat anti-guineapig globulin. Since the anti-rotavirus reagents do not have to be conjugated they can be used at a high dilution (1/6000 to 1/100 000) thus sparing specialised reagents. In addition, because of the amplification effect of using an additional antibody, the colour reaction is stronger, allowing for easier visual identification of positive
specimens. this method we have examined a large number of stools from the United States and Central America for rotavirus. However, when we examined stools from Bangladesh we noted that many gave positive ELISA reactions in the absence of rotavirus. Further investigation revealed that these falsepositive reactions were caused by anti-goat antibodies in the stools, presumably due to the widespread consumption of goat milk. Such anti-goat antibodies could react to the goat serum pre-coat and the goat anti-guineapig globulin, giving falsepositive reactions. This activity can be totally blocked by diluting each of the stools in a solution containing anti-rotavirusnegative goat serum at a 1 % concentration just before putting them into the test. After dilution in goat serum the false-positive reaction disappears, and 183 stools have been successfully studied for the presence of rotavirus. We would also like to report our experience with antisera prepared against antigenically related calf rotavirus. We used high-titred guineapig antisera prepared against two related viruses (Nebraska calf diarrhoea virus and the "0" agent4). These sera could not efficiently detect small amounts of rotavirus in human stools. This was true both for the sandwich and the two-antibody techniques. Possible explanations for this discrepancy include antigenic differences in the viruses used for preparation of the antisera and differences in techniques.
Using
1 Yolken, R. H., Kim, W H , Clem, T., Wyatt, R. G., Kalica, A. R., Chanock, R. M, Kapikian, A. Z. Lancet, 1977, ii, 263. 2 Ellens, D. J., deLeeuw, P. W. ibid. 1977, i, 1363. 3 Yolken, R. H., Greenberg, H , Merson, M., Sack, B., Kapikian, A Z. J. clin. Microbiol.
4 Kapikian, A. Z, Cline,
W. L., Kim, H. W., Kalica, A R., Wyatt, R. G., Van Kirk, D. N, Chanock, R. M., James, H. D. Jr., Vaughn, A. L. Proc. Soc. exp Biol Med. 1976, 152, 535.
We would thus recommend that antisera prepared against human rotavirus- be used unless an antiserum against a related virus has been shown to be effective in efficiently detecting the human rotavirus. Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014, U.S.A.
ROBERT YOLKEN RICHARD G. WYATT ALBERT Z. KAPIKIAN
BONE-MARROW PATTERNS AND CLINICAL STAGING IN CHRONIC LYMPHOCYTE LEUKÆMIA
SIR,-Hernandez-Nieto et al.’ describe a significant association between bone-marrow patterns and the clinical stage of chronic lymphocytic leukaemia (C.L.L.) as classified by Rai et al.2 Since 1974, we have classified c.L.L. patients by a 5-group anatomicoclinical staging system and its prognostic significance was demonstrated in a series of 129 patients.3.4 Our system differs from Rai’s in two respects; patients with spleen but no lymph node involvement are considered as a separate group (stage II) and patients with either anaemia or thrombocytopenia are considered as a single group (stage iv) since we and others5 find them to have a similar median survival (23
months). In 95 bone-marrow
biopsies done early in c.L.L., we found association between the infiltration pattern (diffuse or nodular) and disease stage (see table). an
BONE-MARROW INFILTRATION PATTERN AND CLINICAL STAGE
p
SOAD
TABAQCHALI
JANET K. STEVENS
Community Health Services, City and Hackney Health District, London E9
C. GAZIDIS
ELISA FOR ROTAVIRUS
SIR,-Since the publication of ou’ article on an enzymelinked immunosorbent assay (ELISA)1 we have received many requests for anti-rotavirus sera. To generate a large volume of this reagent, we immunised and obtained sera from a goat. While this serum can be conjugated and used as in our original technique better results are obtained with a two-antibody ELISA similar to that used in our detection system for heatlabile Escherichia coli toxin.2 After pretreatment of wells with goat anti-rotavirus antibody diluted 1/100 000 and addition of the stool specimen to be tested, an unlabelled guineapig antirotavirus antibody diluted 1/6000 is added, followed by an alkaline-phosphatase-labelled goat anti-guineapig globulin. Since the anti-rotavirus reagents do not have to be conjugated they can be used at a high dilution (1/6000 to 1/100 000) thus sparing specialised reagents. In addition, because of the amplification effect of using an additional antibody, the colour reaction is stronger, allowing for easier visual identification of positive
specimens. this method we have examined a large number of stools from the United States and Central America for rotavirus. However, when we examined stools from Bangladesh we noted that many gave positive ELISA reactions in the absence of rotavirus. Further investigation revealed that these falsepositive reactions were caused by anti-goat antibodies in the stools, presumably due to the widespread consumption of goat milk. Such anti-goat antibodies could react to the goat serum pre-coat and the goat anti-guineapig globulin, giving falsepositive reactions. This activity can be totally blocked by diluting each of the stools in a solution containing anti-rotavirusnegative goat serum at a 1 % concentration just before putting them into the test. After dilution in goat serum the false-positive reaction disappears, and 183 stools have been successfully studied for the presence of rotavirus. We would also like to report our experience with antisera prepared against antigenically related calf rotavirus. We used high-titred guineapig antisera prepared against two related viruses (Nebraska calf diarrhoea virus and the "0" agent4). These sera could not efficiently detect small amounts of rotavirus in human stools. This was true both for the sandwich and the two-antibody techniques. Possible explanations for this discrepancy include antigenic differences in the viruses used for preparation of the antisera and differences in techniques.
Using
1 Yolken, R. H., Kim, W H , Clem, T., Wyatt, R. G., Kalica, A. R., Chanock, R. M, Kapikian, A. Z. Lancet, 1977, ii, 263. 2 Ellens, D. J., deLeeuw, P. W. ibid. 1977, i, 1363. 3 Yolken, R. H., Greenberg, H , Merson, M., Sack, B., Kapikian, A Z. J. clin. Microbiol.
4 Kapikian, A. Z, Cline,
W. L., Kim, H. W., Kalica, A R., Wyatt, R. G., Van Kirk, D. N, Chanock, R. M., James, H. D. Jr., Vaughn, A. L. Proc. Soc. exp Biol Med. 1976, 152, 535.
We would thus recommend that antisera prepared against human rotavirus- be used unless an antiserum against a related virus has been shown to be effective in efficiently detecting the human rotavirus. Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014, U.S.A.
ROBERT YOLKEN RICHARD G. WYATT ALBERT Z. KAPIKIAN
BONE-MARROW PATTERNS AND CLINICAL STAGING IN CHRONIC LYMPHOCYTE LEUKÆMIA
SIR,-Hernandez-Nieto et al.’ describe a significant association between bone-marrow patterns and the clinical stage of chronic lymphocytic leukaemia (C.L.L.) as classified by Rai et al.2 Since 1974, we have classified c.L.L. patients by a 5-group anatomicoclinical staging system and its prognostic significance was demonstrated in a series of 129 patients.3.4 Our system differs from Rai’s in two respects; patients with spleen but no lymph node involvement are considered as a separate group (stage II) and patients with either anaemia or thrombocytopenia are considered as a single group (stage iv) since we and others5 find them to have a similar median survival (23
months). In 95 bone-marrow
biopsies done early in c.L.L., we found association between the infiltration pattern (diffuse or nodular) and disease stage (see table). an
BONE-MARROW INFILTRATION PATTERN AND CLINICAL STAGE
p
