1089
biological liver disease at
3 months of age, which
was
followed
by the elimination of HBsAg and by the development of HBsAb. Both mothers fully recovered, with complete disappearance of HBsAg. No anti-HBsAb has occurred in mothers up to 10 and 11 months after hepatitis. From these observations, several points can be made: (1) as reported,’ newborns infected at birth are not always prone to become HBsAg chronic carriers; (2) the maternofetal contamination was likely to have been transplacental, since the babies were born by caesarean section and immediately separated from their mother; (3) the contamination of the babies points to the lack of efficient anti-viral antibodies of maternal origin. Thus, if massive liver necrosis in the mothers was due to a
hyperimmune response, as proposed by Dudley et aI.,2 the contamination and the clinical course in the babies strongly suggest that such a mechanism would not be antibody-mediated. Centre d’Hémobiologie Périnatale, 53 boulevard Diderot, F 75012 Paris. Centre National de Transfusion
Sanguine,
F 91400 Orsay.
I.N.S.E.R.M., Laboratoire d’Immunologie, Hôpital d’Enfants, F 94270 Bicétre.
P. GIRAUD
to learn the outcome of Dr Boxall’s special group of cord-blood-negative babies with breast-milk-positive mothers. We hope that she will draw her conclusions based on the HBsAg outcome of infant’s unconcentrated serum. Our guess is that a baby may occasionally be infected by breast milk but that this is not an important mechanism of transmission.
terested
Department of Epidemiology and Clinical Investigation, U.S. Naval Medical Research Unit no. 2, Box 14, APO San Francisco 96263.
R. PALMER BEASLEY
Department of Epidemiology and International Health, University of Washington, Seattle, Washington; and
CLADD E. STEVENS
Center for Maternal and Child Health, Veterans’ General Hospital, Taipei, Taiwan.
I-SEN SHIAO HSIEN-CHIEH MENG
J. DROUET IDENTIFICATION OF HEPATITIS-B ANTIGEN
J.
M. DUPUY
BREAST-FEEDING AND HEPATITIS B
SIR,-May we reply to Dr Boxall (Nov. 15, p. 979)? The data we presented showed no correlation between breast-feeding and transmission of HBsAg from carrier mothers to their babies. This conclusion requires no assumptions about the presence or absence of HBsAg in breast milk. We were unable to detect HBsAg in unconcentrated breast-milk specimens from carrier women. Dr Boxall reports finding it in 56% of 27 concentrated specimens. Our data simply say that present or absent breast-feeding is not an important means of transmission from mother to child. It seems likely to us that very small amounts of HBsAg detected by Dr Boxall’s highly sensitive methods are not infectious. The fact remains that transmission from asymptomatic carrier mothers to their babies occurs much more frequently in Chinese than Caucasians (it is interesting and undoubtedly biologically significant that Dr Boxall’s only carrier baby is Chinese). Neither Dr Boxall nor most other investigators have commented on the proportion of the babies in their series which were breast-fed. Again, unless the vast majority are not breast-fed, this would suggest that breast-feeding is not an important means of transmission irrespective of the presence of small quantities of HBsAg in the milk of many carrier mothers. Dr Boxall correctly points out that cord-blood-positive infants are probably already infected and thus would not be "susceptible" to post-delivery infection. In our series 21% of the cord bloods were positive and virtually all these infants became carriers. Because there is controversy about the biological significance of a HBsAg-positive cord blood (transplacental infection v. contamination) and because the conclusions were unchanged by making this separation, we chose to present the results for the group without distinction as to cordblood status. The frequency of antigen positivity in cordblood-negative infants was: breast-fed 17/44 (39%); non-breast-fed 14/30 (47%). The p value is 0.5 by Fisher’s exact test. Again Dr Boxall finds a higher frequency of HBsAgpositive cord bloods by concentrating the specimens (57%), but since she has found only 1 baby who became a carrier, there again seems to be little biological meaning to this increased sensitivity. We contend that, when looking at postnatal factors, one should consider excluding only infants who are cordblood-positive and become chronic carriers. We will be in1. Dupuy, J. M., Frommel, D., Alagile, D. Lancet, 1975, i, 191. 2 Dudley, F. J., Fox, R. A., Sherlock, S. ibid. 1972, i, 723.
SiR,—The histological diagnosis of the viral nature of lesions identified in liver biopsies has always presented problems, especially to the busy histopathologist of a district general hospital with little access to immunoflurescence and electron-micoscopy facilities. Recently great hopes have been raised by the demonstration of hepatitis-B antigen (HBAg) in conventional paraffin sections of liver biopsies, using one or the other of Shikata’s staining methods.1-3 Orcein staining appears to be more specific (although the histochemical reaction involved is far from clear), and orcein-positive material seen in the cytoplasm of hepatocytes is thought to represent surface components of HBAg in excess endoplasmic reticulum.4 It has also been shown that positive Shikata staining can be obtained in sections taken from stored paraffin blocks (of liver tissue) up to 50 years of age. Reports so far indicate that orcein staining yields more positive results in biopsy material from seropositive patients with chronic liver disease than from those with acute viral hepatitis. We have reviewed 213 liver biopsies from our files representing material reported between 1964 and 1975 and covering a wide spectrum of liver diseases. Additional sections were cut from all 213 paraffin blocks and stained by a modification of Shikata’s orcein staining method; this modification compares successfully with the original method and presents technical
advantages. Results from
our
retrospective study have shown:
(1) Out of a total of 213 biopsies (including, particularly, 61 cases of cirrhotic changes, 6 cases of chronic persistent hepatitis, and 11 cases of chronic aggressive hepatitis), only 3 biopsies contained orceinpositive material. 2 of these were from 1968 and 1969 when serology for HBAg was not available. The 3rd biopsy came from a patient with positive HBAg but negative antibody. (2) On the other hand, no orcein-positive material could be demonstrated in the biopsies from 5 patients with a positive serology for HBAg and showing histologically chronic liver disease. results may be partly explained by a sampling error-i.e., orcein-positive material could be irregularly distributed in biopsy material. However, we feel that our experience casts some doubt on any over-reliance by histopathologists on these methods. Such histochemical diagnosis of the presence or absence of HBAg in liver biopsies could be deceptive if light microscopy only is used. There is a need for larger prospective studies in which the results obtained with the Shikata’s Some of
our
1. Shikara, T., Uzawa, T., Yoshiwara, N., Akatsuka, T., Yamazaki, S. Jap. J. exp. Med. 1974, 44, 25. 2. Deodhar, K. P., Tapp, E., Scheuer, P. J. J. clin. Path. 1975, 28, 66. 3. Vogel, H. M., Henning, H., Luders, C. J., Braun, H. H., Tripatzis, I Z. Gastroent. 1974, 12, 257. 4. Popper, H. Human Path. 1975, 6, 517.
1090 methods are checked against and correlated with immunofluorescent and electron-microscopic findings. Queen Mary’s Hospital, Roehampton, London, SW
W. V. BOGOMOLETZ MRS D. TUTTLE
5PN.
POMPIDOU’S REFRINGENCE PHENOMENON AND CELL DIFFERENTIATION
SIR,-We demonstrated that the refringence phenomenon restricted to T lymphocytes, but only reflected one of the possible states of the nucleus of any kind of lymphoid cell.’1 Pompidou, who in 1971 had postulated that the refringence phenomenon was typical of T cells,2 revised his previous opinion and suggested that the phenomenon only indicated cell was not
differentiation.3 To check this suggestion
we
counted the percentage of cells
containing non-refringent, slightly refringent, and highly
re-
TOXICITY OF INTRAVENOUS CORYNEBACTERIUM PARVUM
SIR,-We describe here
our
preliminary toxicology experi-
with Corynebacterium parvum given intravenously to patients with solid tumours. We planned to try adjuvant immunotherapy in disseminated disease and hoped that this route would increase the contact between agent and tumour cells and induce optimum effects on the immune and reticuloendothelial systems. Repeated weekly treatments were investigated because long-term outpatient treatment was planned. Intravenous C. parvum (Burroughs Wellcome) was given without other anticancer therapy to twenty-five patients by rapid injection or by infusion of 0 -mg or 2.55 mg per m2 given over 1 hour with premedication. At first patients (group A) were given 2.5 mg/m2 in 20-25 ml of 5% dextrose in water over 20 minutes or less. All had shaking chills and high fevers, starting about an hour after treatment, and then intermittently for 3-6 days. Two had severe nausea and vomiting and refused further treatment. Smaller dosages, greater dilutions, longer infusions, and prophylactic paracetamol (acetaminophen) and prochlorperaence
REACTIONS TO INTRAVENOUS C. PARVUM
A: C. parvum C2’5 mg/m2) diluted in 20-250 ml of 5% dextrose and given over 20 minutes or less. Group B C. parvum (B,0t, B2 2.5mg/m2) in 250 ml of 5% dextrose and water or normal sahne given over one hour. All patients in group B received prophvlactic acetaminophen and prochlorperazine at time of infusion.
Group water
in immunoblasts (LB.), immature plasma-cells (I.P.), and mature plasma-cells (M.P.). N=non-refringent. S=slightly refringent. H=highly refringent.
Refringence
fringent nuclei in immunoblasts, in immature plasma-cells, and in mature plasma-cells. The sources were as follows: (1) axillary lymph-nodes from five rabbits with a clear plasma-cell reaction and high percentage of mature plasma-cells after five days of acute appendicitis and peritonitis; (2) axillary lymphnodes from five CBA mice after eight days of immunisation with Vi-antigen of typhoid organisms, when numerous immature plasma-cells were present. Immunoblasts, immature plasma-cells, and mature plasmaall stages of differentiation of the
cell range and The differences in distinguish morphologically. numbers of non-refringent, slightly refringent, and highly-refringent nuclei in these groups of cells (see accompanying figure) lead us to suggest that non-refringent nuclei are characteristic of immature cells, low refringence indicates the start of differentiation, and high refringence is typical of highly differentiated cells. We never saw the refringence phenomenon in germinal centres and in human lymphosarcoma cells.
cells are
are
same
easy to
Transplantation, 55th City Hospital, 2nd Zagorodny Pereulok 18A, Building 4, 113152 Moscow, U.S.S.R.
DINA P. LINDNER ISOLDA A. POBERIY
OLGA N. STETZENKO
1. Lindner, D. P., Pobeny, I. A. Lancet, 1974, ii, 724. 2. Pompidou, A., Schramm, B. ibid. 1971, ii, 245. 3. Pompidou, A., Contesso, G. Path. Biol., Paris, 1974,
zine were tried. The table compares group A with group B patients who received as their first treatment either 0.1 or 2.5 mg/m2 in 250 ml 5% dextrose and water or in physiological saline given over 1 hour. Reactions were less common and lasted for a shorter time (usually less than 12 hours) in
group-B patients. Repeated weekly treatments were associated with decreasing reactions. Nine patients were given 3-12 weekly injections of 1-2.5mg/m2 of C. parvum intravenously by various schedules of administration. By the second or third treatments only mild reactions lasting less than 12 hours were encountered. By this stage of treatment, more rapid administration (10 minutes) and smaller amounts of diluent (20 ml), with doses up to 2.5 mg/m2, did not increase the side-effects. Burroughs Well come have on record occasional cases of acute blood-pressure alterations during intravenous administration.’ The fifteen patients receiving one-hour injections were monitored every 15 minutes and demonstrated a high of 144/100 mm Hg and a low of 100/55 mm Hg with systolic and diastolic levels demonstrating less than 20% changes from pretreatment levels. Renal, liver, and metabolic status were monitored, especially in nine patients receiving multiple weekly infusions. Three out of nine had elevations in their serum-alkaline-phosphatase. There was a transient fall in lymphocytes, monocytes, and platelets at 24 hours after treatment, but these were back to pre-treatment levels at one week. Modifying the intravenous administration of C. parvum by increasing the duration of infusion during the first two or three treatments plus premedication allows a clinically well-tolerated adminstration of up to 2.5 mg/m2 of C. parvum on a weekly basis. Close monitoring of vital signs is recommended because 1.
22,
143
R. M., Cochran, A. J., C. Lancet, 1974, ii, 1096.
Grant,
Hoyle, D., Mackie, R., Murray, E. L., Ross.
biological liver disease at
3 months of age, which
was
followed
by the elimination of HBsAg and by the development of HBsAb. Both mothers fully recovered, with complete disappearance of HBsAg. No anti-HBsAb has occurred in mothers up to 10 and 11 months after hepatitis. From these observations, several points can be made: (1) as reported,’ newborns infected at birth are not always prone to become HBsAg chronic carriers; (2) the maternofetal contamination was likely to have been transplacental, since the babies were born by caesarean section and immediately separated from their mother; (3) the contamination of the babies points to the lack of efficient anti-viral antibodies of maternal origin. Thus, if massive liver necrosis in the mothers was due to a
hyperimmune response, as proposed by Dudley et aI.,2 the contamination and the clinical course in the babies strongly suggest that such a mechanism would not be antibody-mediated. Centre d’Hémobiologie Périnatale, 53 boulevard Diderot, F 75012 Paris. Centre National de Transfusion
Sanguine,
F 91400 Orsay.
I.N.S.E.R.M., Laboratoire d’Immunologie, Hôpital d’Enfants, F 94270 Bicétre.
P. GIRAUD
to learn the outcome of Dr Boxall’s special group of cord-blood-negative babies with breast-milk-positive mothers. We hope that she will draw her conclusions based on the HBsAg outcome of infant’s unconcentrated serum. Our guess is that a baby may occasionally be infected by breast milk but that this is not an important mechanism of transmission.
terested
Department of Epidemiology and Clinical Investigation, U.S. Naval Medical Research Unit no. 2, Box 14, APO San Francisco 96263.
R. PALMER BEASLEY
Department of Epidemiology and International Health, University of Washington, Seattle, Washington; and
CLADD E. STEVENS
Center for Maternal and Child Health, Veterans’ General Hospital, Taipei, Taiwan.
I-SEN SHIAO HSIEN-CHIEH MENG
J. DROUET IDENTIFICATION OF HEPATITIS-B ANTIGEN
J.
M. DUPUY
BREAST-FEEDING AND HEPATITIS B
SIR,-May we reply to Dr Boxall (Nov. 15, p. 979)? The data we presented showed no correlation between breast-feeding and transmission of HBsAg from carrier mothers to their babies. This conclusion requires no assumptions about the presence or absence of HBsAg in breast milk. We were unable to detect HBsAg in unconcentrated breast-milk specimens from carrier women. Dr Boxall reports finding it in 56% of 27 concentrated specimens. Our data simply say that present or absent breast-feeding is not an important means of transmission from mother to child. It seems likely to us that very small amounts of HBsAg detected by Dr Boxall’s highly sensitive methods are not infectious. The fact remains that transmission from asymptomatic carrier mothers to their babies occurs much more frequently in Chinese than Caucasians (it is interesting and undoubtedly biologically significant that Dr Boxall’s only carrier baby is Chinese). Neither Dr Boxall nor most other investigators have commented on the proportion of the babies in their series which were breast-fed. Again, unless the vast majority are not breast-fed, this would suggest that breast-feeding is not an important means of transmission irrespective of the presence of small quantities of HBsAg in the milk of many carrier mothers. Dr Boxall correctly points out that cord-blood-positive infants are probably already infected and thus would not be "susceptible" to post-delivery infection. In our series 21% of the cord bloods were positive and virtually all these infants became carriers. Because there is controversy about the biological significance of a HBsAg-positive cord blood (transplacental infection v. contamination) and because the conclusions were unchanged by making this separation, we chose to present the results for the group without distinction as to cordblood status. The frequency of antigen positivity in cordblood-negative infants was: breast-fed 17/44 (39%); non-breast-fed 14/30 (47%). The p value is 0.5 by Fisher’s exact test. Again Dr Boxall finds a higher frequency of HBsAgpositive cord bloods by concentrating the specimens (57%), but since she has found only 1 baby who became a carrier, there again seems to be little biological meaning to this increased sensitivity. We contend that, when looking at postnatal factors, one should consider excluding only infants who are cordblood-positive and become chronic carriers. We will be in1. Dupuy, J. M., Frommel, D., Alagile, D. Lancet, 1975, i, 191. 2 Dudley, F. J., Fox, R. A., Sherlock, S. ibid. 1972, i, 723.
SiR,—The histological diagnosis of the viral nature of lesions identified in liver biopsies has always presented problems, especially to the busy histopathologist of a district general hospital with little access to immunoflurescence and electron-micoscopy facilities. Recently great hopes have been raised by the demonstration of hepatitis-B antigen (HBAg) in conventional paraffin sections of liver biopsies, using one or the other of Shikata’s staining methods.1-3 Orcein staining appears to be more specific (although the histochemical reaction involved is far from clear), and orcein-positive material seen in the cytoplasm of hepatocytes is thought to represent surface components of HBAg in excess endoplasmic reticulum.4 It has also been shown that positive Shikata staining can be obtained in sections taken from stored paraffin blocks (of liver tissue) up to 50 years of age. Reports so far indicate that orcein staining yields more positive results in biopsy material from seropositive patients with chronic liver disease than from those with acute viral hepatitis. We have reviewed 213 liver biopsies from our files representing material reported between 1964 and 1975 and covering a wide spectrum of liver diseases. Additional sections were cut from all 213 paraffin blocks and stained by a modification of Shikata’s orcein staining method; this modification compares successfully with the original method and presents technical
advantages. Results from
our
retrospective study have shown:
(1) Out of a total of 213 biopsies (including, particularly, 61 cases of cirrhotic changes, 6 cases of chronic persistent hepatitis, and 11 cases of chronic aggressive hepatitis), only 3 biopsies contained orceinpositive material. 2 of these were from 1968 and 1969 when serology for HBAg was not available. The 3rd biopsy came from a patient with positive HBAg but negative antibody. (2) On the other hand, no orcein-positive material could be demonstrated in the biopsies from 5 patients with a positive serology for HBAg and showing histologically chronic liver disease. results may be partly explained by a sampling error-i.e., orcein-positive material could be irregularly distributed in biopsy material. However, we feel that our experience casts some doubt on any over-reliance by histopathologists on these methods. Such histochemical diagnosis of the presence or absence of HBAg in liver biopsies could be deceptive if light microscopy only is used. There is a need for larger prospective studies in which the results obtained with the Shikata’s Some of
our
1. Shikara, T., Uzawa, T., Yoshiwara, N., Akatsuka, T., Yamazaki, S. Jap. J. exp. Med. 1974, 44, 25. 2. Deodhar, K. P., Tapp, E., Scheuer, P. J. J. clin. Path. 1975, 28, 66. 3. Vogel, H. M., Henning, H., Luders, C. J., Braun, H. H., Tripatzis, I Z. Gastroent. 1974, 12, 257. 4. Popper, H. Human Path. 1975, 6, 517.
1090 methods are checked against and correlated with immunofluorescent and electron-microscopic findings. Queen Mary’s Hospital, Roehampton, London, SW
W. V. BOGOMOLETZ MRS D. TUTTLE
5PN.
POMPIDOU’S REFRINGENCE PHENOMENON AND CELL DIFFERENTIATION
SIR,-We demonstrated that the refringence phenomenon restricted to T lymphocytes, but only reflected one of the possible states of the nucleus of any kind of lymphoid cell.’1 Pompidou, who in 1971 had postulated that the refringence phenomenon was typical of T cells,2 revised his previous opinion and suggested that the phenomenon only indicated cell was not
differentiation.3 To check this suggestion
we
counted the percentage of cells
containing non-refringent, slightly refringent, and highly
re-
TOXICITY OF INTRAVENOUS CORYNEBACTERIUM PARVUM
SIR,-We describe here
our
preliminary toxicology experi-
with Corynebacterium parvum given intravenously to patients with solid tumours. We planned to try adjuvant immunotherapy in disseminated disease and hoped that this route would increase the contact between agent and tumour cells and induce optimum effects on the immune and reticuloendothelial systems. Repeated weekly treatments were investigated because long-term outpatient treatment was planned. Intravenous C. parvum (Burroughs Wellcome) was given without other anticancer therapy to twenty-five patients by rapid injection or by infusion of 0 -mg or 2.55 mg per m2 given over 1 hour with premedication. At first patients (group A) were given 2.5 mg/m2 in 20-25 ml of 5% dextrose in water over 20 minutes or less. All had shaking chills and high fevers, starting about an hour after treatment, and then intermittently for 3-6 days. Two had severe nausea and vomiting and refused further treatment. Smaller dosages, greater dilutions, longer infusions, and prophylactic paracetamol (acetaminophen) and prochlorperaence
REACTIONS TO INTRAVENOUS C. PARVUM
A: C. parvum C2’5 mg/m2) diluted in 20-250 ml of 5% dextrose and given over 20 minutes or less. Group B C. parvum (B,0t, B2 2.5mg/m2) in 250 ml of 5% dextrose and water or normal sahne given over one hour. All patients in group B received prophvlactic acetaminophen and prochlorperazine at time of infusion.
Group water
in immunoblasts (LB.), immature plasma-cells (I.P.), and mature plasma-cells (M.P.). N=non-refringent. S=slightly refringent. H=highly refringent.
Refringence
fringent nuclei in immunoblasts, in immature plasma-cells, and in mature plasma-cells. The sources were as follows: (1) axillary lymph-nodes from five rabbits with a clear plasma-cell reaction and high percentage of mature plasma-cells after five days of acute appendicitis and peritonitis; (2) axillary lymphnodes from five CBA mice after eight days of immunisation with Vi-antigen of typhoid organisms, when numerous immature plasma-cells were present. Immunoblasts, immature plasma-cells, and mature plasmaall stages of differentiation of the
cell range and The differences in distinguish morphologically. numbers of non-refringent, slightly refringent, and highly-refringent nuclei in these groups of cells (see accompanying figure) lead us to suggest that non-refringent nuclei are characteristic of immature cells, low refringence indicates the start of differentiation, and high refringence is typical of highly differentiated cells. We never saw the refringence phenomenon in germinal centres and in human lymphosarcoma cells.
cells are
are
same
easy to
Transplantation, 55th City Hospital, 2nd Zagorodny Pereulok 18A, Building 4, 113152 Moscow, U.S.S.R.
DINA P. LINDNER ISOLDA A. POBERIY
OLGA N. STETZENKO
1. Lindner, D. P., Pobeny, I. A. Lancet, 1974, ii, 724. 2. Pompidou, A., Schramm, B. ibid. 1971, ii, 245. 3. Pompidou, A., Contesso, G. Path. Biol., Paris, 1974,
zine were tried. The table compares group A with group B patients who received as their first treatment either 0.1 or 2.5 mg/m2 in 250 ml 5% dextrose and water or in physiological saline given over 1 hour. Reactions were less common and lasted for a shorter time (usually less than 12 hours) in
group-B patients. Repeated weekly treatments were associated with decreasing reactions. Nine patients were given 3-12 weekly injections of 1-2.5mg/m2 of C. parvum intravenously by various schedules of administration. By the second or third treatments only mild reactions lasting less than 12 hours were encountered. By this stage of treatment, more rapid administration (10 minutes) and smaller amounts of diluent (20 ml), with doses up to 2.5 mg/m2, did not increase the side-effects. Burroughs Well come have on record occasional cases of acute blood-pressure alterations during intravenous administration.’ The fifteen patients receiving one-hour injections were monitored every 15 minutes and demonstrated a high of 144/100 mm Hg and a low of 100/55 mm Hg with systolic and diastolic levels demonstrating less than 20% changes from pretreatment levels. Renal, liver, and metabolic status were monitored, especially in nine patients receiving multiple weekly infusions. Three out of nine had elevations in their serum-alkaline-phosphatase. There was a transient fall in lymphocytes, monocytes, and platelets at 24 hours after treatment, but these were back to pre-treatment levels at one week. Modifying the intravenous administration of C. parvum by increasing the duration of infusion during the first two or three treatments plus premedication allows a clinically well-tolerated adminstration of up to 2.5 mg/m2 of C. parvum on a weekly basis. Close monitoring of vital signs is recommended because 1.
22,
143
R. M., Cochran, A. J., C. Lancet, 1974, ii, 1096.
Grant,
Hoyle, D., Mackie, R., Murray, E. L., Ross.
