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TEST FOR BILIRUBIN IN LIQUOR AMNII
1210 milk. These swallowed fluids were seen in the trachea after deflation of the cuff and were sucked out. When the patient was breathing spontaneously for short periods a swallowing test was performed, with the same result. It was thought, therefore, that despite the measures described, an cesophagotracheal fistula, due to pressure necrosis, had developed, but this diagnosis was not verified by bronchoscopy and cesophagoscopy-only severe inflammation of the upper trachea was seen. A silver tracheostomy tube was inserted, but did not improve swallowing. Intravenous feeding was kept up for 2 more days. By then the patient’s respiration was adequate and the tracheostomy could be closed. Thereafter the patient was able to swallow normally and no further aspiration occurred. Her lungs stayed clear and 2 weeks later she was sent home.
know whether tracheal ballooning occurred in this but I agree with the points made by Dr. Feldman and patient, his colleagues in their discussion. In my patient the disturbance of the swallowing movement because of tracheal fixation was probably the most important point. University Surgical Clinic, W. F. LIST. Graz, Austria. I do
not
since it may be associated with extensive damage to gastrointestinal smooth muscle. 12 normal sera and purified samples of IgA, IgG, and IgM immunoglobulins were also tested. Brilliant staining of gastrointestinal and vascular smooth muscle of rat and human origin was obtained with lupoid-
hepatitis sera only. Using fluorescein-conjugated anti-IgA, anti-IgG, and anti-IgM for tracing, positive results were obtained only with anti-IgG and lupoid-hepatitis sera. Fractionation of these sera into 3 parts by gel filtration through Sephadex G200 ’ showed that the staining properties were confined to the fraction containing the 7S-globulins. Staining was inhibited by absorption of the sera with homogenates of ’
stomach or liver or human kidney, but not by human AB red cells. Apart from staining of smooth muscle, we have the impression that stromal elements including capillary walls (e.g., in renal glomeruli) react specifically with the antisera, but found no other convincing staining reaction in skeletal or cardiac muscle, brain, kidney, liver, or adrenal. The vascular tissue in liver or kidney homogenates could be responsible for their effectiveness in serum-absorption. Possible identity of the antibody concerned with the complement-fixing antibody demonstrated in high titre in lupoid
rat or
sheep
hepatitis5 requires investigation. TEST FOR BILIRUBIN IN LIQUOR AMNII SIR,-In the second paragraph of their article (May 7) Mr. Bower and Dr. Swale state: " The second group make use of the diazo reaction to estimate the amount of bilirubin present (Wild 1961, Watson 1962, Pennington and Hall 1966)." We would point out that our method, which, they cite, does not employ a diazo reaction and is dependent upon the solubility of indirect bilirubin in chloroform and its subsequent examination spectrophotometrically at 450 m-. The sensitivity of our method is extremely high, in contradistinction to the many methods which employ the diazo reaction; in this reaction the addition of various chemical solutions brings about a pronounced dilution of the specimen under examination. The method which we have described brings about a fourfold concentration of bilirubin, and has a sensitivity which permits less than 0-05 mg. per 100 ml. of bilirubin to be detected. Using this method a large number of cases of Rh-negative pregnant women have been investigated ; predictions have been made on the foetal condition, with very encouragining results. We hope to publish these results when their numbers allow a thorough analysis to be made. Sheffield and Region Endocrine Investigation Centre, G. W. PENNINGTON Jessop Hospital for Women, R. HALL. Sheffield 3
SMOOTH-MUSCLE ANTIBODY IN LUPOID HEPATITIS SIR,-We should like to congratulate Mr. Johnson and his co-workers2 on their application of our immunofluorescence method of detecting pernicious-ansemia autoantibodies3 to the recognition of an antibody to smooth muscle in the sera of patients with lupoid hepatitis.- Following their procedure we have confirmed their observation with 4 lupoid-hepatitis sera (kindly provided by Dr. 1. R. Mackay), and have further investigated the significance of their results. To exclude one possible source of non-specific reaction to the high serum-globulin levels in lupoid hepatitis, we examined sera from patients with diseases in which there is a selective increase in one or more of the immunoglobulins-i.e., myeloma of IgG type in 3 patients and of IgA type in 1 patient, follicular lymphoma with macroglobulinxmia in 1 patient, rheumatoid arthritis in 3 patients, and scleroderma in 4 patients. The investigation of scleroderma is perhaps especially appropriate 1. Pennington, G. W., Hall, R. J. clin. Path. 1966, 19, 90. 2. Johnson, G. D., Holborow, E. J., Glynn, L. E. Lancet, 1965, ii, 878. 3. de Boer, W. G. R. M., Nairn, R. C., Maxwell, A. J. clin. Path. 1965, 18, 456. 4. Mackay, I. R., Burnet, F. M. Autoimmune Diseases. Springfield, Illinois, 1963.
Department of Pathology, Monash University, Melbourne, Australia.
P.
N. J. IRONSIDE W. G. R. M. DE BOER R. C. NAIRN.
DIAGNOSING PRIMARY BILIARY CIRRHOSIS
SiR,ņWe want to emphasise the great interest of the observation made by Walker et all that " the serum of patients with primary biliary cirrhosis contains antibodies which give a distinctive pattern of cytoplasmic staining in tissue sections ". We have investigated a 60-year-old housewife who developed intense pruritus in 1962. In September, 1965, jaundice, hepatomegaly, and cholecystic pain were noted. Laparotomy
Stained section of rat kidney showing unstained glomerulus (G), and fluorescence of tubular cells. (Reduced to about threequarters from x 400.)
and cholangiography in February, 1966, showed no obstruction of main bileducts. On admission to the department of hepatology of this institute, severe jaundice, hypercholestersmia, and high alkaline phosphatase were observed. Needle liver-biopsy was consistent with a diagnosis of biliary cirrhosis. We looked for cytoplasmic antibodies in the serum of the patient, diluted 1/10, by fluorescent test, using the double-layer technique. Tests were performed with fluorescein-conjugated rabbit anti-7S-human-y-globulin, using fixed cryostat sections of rat kidney. On microscopy striking fluorescence of tubular cells was noted; glomeruli were unstained (see accompanying 5. 6.
Gajdusek, D. C. Archs intern. Med. 1958, 101, 9. Walker, J. G., Doniach, D., Roitt, I. M., Sherlock, S. Lancet, 1965, i, 827.
know whether tracheal ballooning occurred in this but I agree with the points made by Dr. Feldman and patient, his colleagues in their discussion. In my patient the disturbance of the swallowing movement because of tracheal fixation was probably the most important point. University Surgical Clinic, W. F. LIST. Graz, Austria. I do
not
since it may be associated with extensive damage to gastrointestinal smooth muscle. 12 normal sera and purified samples of IgA, IgG, and IgM immunoglobulins were also tested. Brilliant staining of gastrointestinal and vascular smooth muscle of rat and human origin was obtained with lupoid-
hepatitis sera only. Using fluorescein-conjugated anti-IgA, anti-IgG, and anti-IgM for tracing, positive results were obtained only with anti-IgG and lupoid-hepatitis sera. Fractionation of these sera into 3 parts by gel filtration through Sephadex G200 ’ showed that the staining properties were confined to the fraction containing the 7S-globulins. Staining was inhibited by absorption of the sera with homogenates of ’
stomach or liver or human kidney, but not by human AB red cells. Apart from staining of smooth muscle, we have the impression that stromal elements including capillary walls (e.g., in renal glomeruli) react specifically with the antisera, but found no other convincing staining reaction in skeletal or cardiac muscle, brain, kidney, liver, or adrenal. The vascular tissue in liver or kidney homogenates could be responsible for their effectiveness in serum-absorption. Possible identity of the antibody concerned with the complement-fixing antibody demonstrated in high titre in lupoid
rat or
sheep
hepatitis5 requires investigation. TEST FOR BILIRUBIN IN LIQUOR AMNII SIR,-In the second paragraph of their article (May 7) Mr. Bower and Dr. Swale state: " The second group make use of the diazo reaction to estimate the amount of bilirubin present (Wild 1961, Watson 1962, Pennington and Hall 1966)." We would point out that our method, which, they cite, does not employ a diazo reaction and is dependent upon the solubility of indirect bilirubin in chloroform and its subsequent examination spectrophotometrically at 450 m-. The sensitivity of our method is extremely high, in contradistinction to the many methods which employ the diazo reaction; in this reaction the addition of various chemical solutions brings about a pronounced dilution of the specimen under examination. The method which we have described brings about a fourfold concentration of bilirubin, and has a sensitivity which permits less than 0-05 mg. per 100 ml. of bilirubin to be detected. Using this method a large number of cases of Rh-negative pregnant women have been investigated ; predictions have been made on the foetal condition, with very encouragining results. We hope to publish these results when their numbers allow a thorough analysis to be made. Sheffield and Region Endocrine Investigation Centre, G. W. PENNINGTON Jessop Hospital for Women, R. HALL. Sheffield 3
SMOOTH-MUSCLE ANTIBODY IN LUPOID HEPATITIS SIR,-We should like to congratulate Mr. Johnson and his co-workers2 on their application of our immunofluorescence method of detecting pernicious-ansemia autoantibodies3 to the recognition of an antibody to smooth muscle in the sera of patients with lupoid hepatitis.- Following their procedure we have confirmed their observation with 4 lupoid-hepatitis sera (kindly provided by Dr. 1. R. Mackay), and have further investigated the significance of their results. To exclude one possible source of non-specific reaction to the high serum-globulin levels in lupoid hepatitis, we examined sera from patients with diseases in which there is a selective increase in one or more of the immunoglobulins-i.e., myeloma of IgG type in 3 patients and of IgA type in 1 patient, follicular lymphoma with macroglobulinxmia in 1 patient, rheumatoid arthritis in 3 patients, and scleroderma in 4 patients. The investigation of scleroderma is perhaps especially appropriate 1. Pennington, G. W., Hall, R. J. clin. Path. 1966, 19, 90. 2. Johnson, G. D., Holborow, E. J., Glynn, L. E. Lancet, 1965, ii, 878. 3. de Boer, W. G. R. M., Nairn, R. C., Maxwell, A. J. clin. Path. 1965, 18, 456. 4. Mackay, I. R., Burnet, F. M. Autoimmune Diseases. Springfield, Illinois, 1963.
Department of Pathology, Monash University, Melbourne, Australia.
P.
N. J. IRONSIDE W. G. R. M. DE BOER R. C. NAIRN.
DIAGNOSING PRIMARY BILIARY CIRRHOSIS
SiR,ņWe want to emphasise the great interest of the observation made by Walker et all that " the serum of patients with primary biliary cirrhosis contains antibodies which give a distinctive pattern of cytoplasmic staining in tissue sections ". We have investigated a 60-year-old housewife who developed intense pruritus in 1962. In September, 1965, jaundice, hepatomegaly, and cholecystic pain were noted. Laparotomy
Stained section of rat kidney showing unstained glomerulus (G), and fluorescence of tubular cells. (Reduced to about threequarters from x 400.)
and cholangiography in February, 1966, showed no obstruction of main bileducts. On admission to the department of hepatology of this institute, severe jaundice, hypercholestersmia, and high alkaline phosphatase were observed. Needle liver-biopsy was consistent with a diagnosis of biliary cirrhosis. We looked for cytoplasmic antibodies in the serum of the patient, diluted 1/10, by fluorescent test, using the double-layer technique. Tests were performed with fluorescein-conjugated rabbit anti-7S-human-y-globulin, using fixed cryostat sections of rat kidney. On microscopy striking fluorescence of tubular cells was noted; glomeruli were unstained (see accompanying 5. 6.
Gajdusek, D. C. Archs intern. Med. 1958, 101, 9. Walker, J. G., Doniach, D., Roitt, I. M., Sherlock, S. Lancet, 1965, i, 827.