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LYMPHOCYTE-TYPING CHANGES AFTER SHORT-TERM CULTURE
262 with previous hepatitis. In addition there was gross vacuolation and fatty change in liver parenchymal cells and scattered bacterial colonies. While previous drug therapy may possibly have been an ,-tiolo 9 ical factor, it seems much more probable that the infectious hepatitis was the significant event, and moreover that the excessive menstrual bleeding was the first manifestation of marrow aplasia. Most patients reported to have this complication of infectious hepatitis have been male,1 males exceeding females by about 4 to 1.22 The fact that aplastic anxmia was related to a second episode of infectious hepatitis is unusual. During pregnancy it is possible to confuse the clinical and biochemical features of infectious hepatitis and benign cholestatic jaundice of pregnancy.3 Without the benefit of liver histology during the pregnancy4 it was impossible to be certain this woman had infectious hepatitis and not obstetric cholestasis, although the evidence was thought strongly in favour of the former diagnosis. Queen Victoria Hospital, Melbourne, Victoria, Australia.
RESULTS OF HL-A TYPING OF LYMPHOCYTES FROM FRESH BLOOD AND FROM SHORT-TERM CULTURE, FROM MEMBERS OF ONE HEALTHY FAMILY
M. LE MOINE PARKER.
LYMPHOCYTE-TYPING CHANGES AFTER SHORT-TERM CULTURE
SIR,-Mrs. Mackintosh and her co-workers5 have
re-
ported the appearance of "new" HL-A antigens on lymphocytes after short-term culture. Dick and Steel have reported similar findings with lymphoblastoid cells in long-term culture.6 We wish to report the results of a study of lymphocytes from members of one family group. Samples from the family members were tested in nine erythrocyte-marker systems, six serum systems, and four erythrocyte enzyme systems. No evidence of illegitimacy was found. The lymphocytes were cultured in Eagle’s M.E.M. with 20% autologous plasma for 4 days. Phytohxmagglutinin (P.H.A.) was used as a stimulant. After culture, the lymphocytes were re-suspended in Eagle’s M.E.M. and typed by means of the micro-lymphocytotoxicity test, using rabbit serum as a source of complement. The results of typing lymphocytes from fresh blood and from P.H.A.-stimulated cultures are shown in the accompanying table.
Only positive test results obtained with antisera believed to be monospecific are included. The presumably monospecific antisera we used reveal the following specificities at the first sublocus: HL-A1, 2, 3, 9, LA-4, Lcl7; and at the second sublocus: HL-A5, 7, 8, 12. All typings were done blindly, and cells from two parallel cultures
were
tested for each individual.
There
was
complete correspondence between the parallel tests. No antigen present on lymphocytes from fresh blood was missing after short-term culture. New " specificities appeared on cultured cells from all "
individuals. The " new " antigens assigned to the first sublocus on cultured cells from the children were all demonstrable on the lymphocytes from one or both parents, either in fresh blood or after short-term culture. The Lcl7 antigen was present also on fresh lymphocytes from the second son, whereas this antigen was demonstrable onlv after short-term culture on cells from the parents and the third and fourth child. Concerning the second sublocus, cells from the first Lancet, 1971, i, 844. Rubin, E., Gottlieb, C., Vogel, P. Am. J. Med. 1968, 45, 88. Bennett, M. McK., Forbes, J. A., Lucas, C. R., Kucers, A. Med. J. Aust. 1967, ii, 974. 4. Parker, M. L. ibid. p. 967. 5. Mackintosh, P., Hardy, D. A., Aviet, T. Lancet, 1971, i, 1019. 6. Dick, H. M., Steel, C. M. ibid. p. 1135. 1. 2. 3.
child exhibited the HL-A12 antigen after short-term culture, whereas this antigen could not be demonstrated on the cells from any of the parents. From a genetic point of view, the results of this study have several interesting aspects. More than two antigens belonging to the first sublocus were expressed after shortTaken at face term culture of cells from all individuals. indicate that the rule that not more than value, these results " two HL-A alleles " can be expressed per " sublocus does not hold true for cells grown in culture. Relatively few monospecific reagents were used in this study. It is possible that other " new " antigens would have been found if additional antisera had been used. Not all antigens which could have been revealed by the reagents were demonstrable on the cells after short-term culture. This, as well as the consistently negative controls, indicates that the appearance of new " antigens on the cells after culture is "
"
not an
entirely non-specific phenomenon.
We find the apparent exception from the presumed dominant inheritance of the Lcl7 antigen notable since the antiserum used is operationally monospecific. The reactions were clear-cut: fresh cells from both parents were negative (10% stained cells) and from the second child clearly positive (80% stained cells). However, the Lcl7 antigen was present on cultured cells from both parents. Mackintosh et al. discuss the possibility that the appearance of " new " antigens on cells in culture could be due to low-titre antibodies of other or cross-reacting specificities in the antisera. This possibility now appears somewhat less likely since we have demonstrated the HL-A12 antigen on the cells from the first child, whereas cells from none of the parents possessed this antigen. At present, we tend to favour the interpretation that new or modified antigens become detectable on lymphocytes after blastoid transformation. These data indicate that further investigation of the expression of HL-A antigens under different conditions are highly needed. If the present results are substantiated by additional studies, they may have important implications for the genetic and molecular concept of the HL-A system. Institute of Medical Genetics, GRETHE NOER University of Oslo, CARL BIRGER VAN DER HAGEN Blindern, Oslo 3, KÅRE BERG. Norway.
RESULTS OF HL-A TYPING OF LYMPHOCYTES FROM FRESH BLOOD AND FROM SHORT-TERM CULTURE, FROM MEMBERS OF ONE HEALTHY FAMILY
M. LE MOINE PARKER.
LYMPHOCYTE-TYPING CHANGES AFTER SHORT-TERM CULTURE
SIR,-Mrs. Mackintosh and her co-workers5 have
re-
ported the appearance of "new" HL-A antigens on lymphocytes after short-term culture. Dick and Steel have reported similar findings with lymphoblastoid cells in long-term culture.6 We wish to report the results of a study of lymphocytes from members of one family group. Samples from the family members were tested in nine erythrocyte-marker systems, six serum systems, and four erythrocyte enzyme systems. No evidence of illegitimacy was found. The lymphocytes were cultured in Eagle’s M.E.M. with 20% autologous plasma for 4 days. Phytohxmagglutinin (P.H.A.) was used as a stimulant. After culture, the lymphocytes were re-suspended in Eagle’s M.E.M. and typed by means of the micro-lymphocytotoxicity test, using rabbit serum as a source of complement. The results of typing lymphocytes from fresh blood and from P.H.A.-stimulated cultures are shown in the accompanying table.
Only positive test results obtained with antisera believed to be monospecific are included. The presumably monospecific antisera we used reveal the following specificities at the first sublocus: HL-A1, 2, 3, 9, LA-4, Lcl7; and at the second sublocus: HL-A5, 7, 8, 12. All typings were done blindly, and cells from two parallel cultures
were
tested for each individual.
There
was
complete correspondence between the parallel tests. No antigen present on lymphocytes from fresh blood was missing after short-term culture. New " specificities appeared on cultured cells from all "
individuals. The " new " antigens assigned to the first sublocus on cultured cells from the children were all demonstrable on the lymphocytes from one or both parents, either in fresh blood or after short-term culture. The Lcl7 antigen was present also on fresh lymphocytes from the second son, whereas this antigen was demonstrable onlv after short-term culture on cells from the parents and the third and fourth child. Concerning the second sublocus, cells from the first Lancet, 1971, i, 844. Rubin, E., Gottlieb, C., Vogel, P. Am. J. Med. 1968, 45, 88. Bennett, M. McK., Forbes, J. A., Lucas, C. R., Kucers, A. Med. J. Aust. 1967, ii, 974. 4. Parker, M. L. ibid. p. 967. 5. Mackintosh, P., Hardy, D. A., Aviet, T. Lancet, 1971, i, 1019. 6. Dick, H. M., Steel, C. M. ibid. p. 1135. 1. 2. 3.
child exhibited the HL-A12 antigen after short-term culture, whereas this antigen could not be demonstrated on the cells from any of the parents. From a genetic point of view, the results of this study have several interesting aspects. More than two antigens belonging to the first sublocus were expressed after shortTaken at face term culture of cells from all individuals. indicate that the rule that not more than value, these results " two HL-A alleles " can be expressed per " sublocus does not hold true for cells grown in culture. Relatively few monospecific reagents were used in this study. It is possible that other " new " antigens would have been found if additional antisera had been used. Not all antigens which could have been revealed by the reagents were demonstrable on the cells after short-term culture. This, as well as the consistently negative controls, indicates that the appearance of new " antigens on the cells after culture is "
"
not an
entirely non-specific phenomenon.
We find the apparent exception from the presumed dominant inheritance of the Lcl7 antigen notable since the antiserum used is operationally monospecific. The reactions were clear-cut: fresh cells from both parents were negative (10% stained cells) and from the second child clearly positive (80% stained cells). However, the Lcl7 antigen was present on cultured cells from both parents. Mackintosh et al. discuss the possibility that the appearance of " new " antigens on cells in culture could be due to low-titre antibodies of other or cross-reacting specificities in the antisera. This possibility now appears somewhat less likely since we have demonstrated the HL-A12 antigen on the cells from the first child, whereas cells from none of the parents possessed this antigen. At present, we tend to favour the interpretation that new or modified antigens become detectable on lymphocytes after blastoid transformation. These data indicate that further investigation of the expression of HL-A antigens under different conditions are highly needed. If the present results are substantiated by additional studies, they may have important implications for the genetic and molecular concept of the HL-A system. Institute of Medical Genetics, GRETHE NOER University of Oslo, CARL BIRGER VAN DER HAGEN Blindern, Oslo 3, KÅRE BERG. Norway.