- Email: [email protected]
PREVENTION OF RECURRENT ABORTION WITH LEUCOCYTE TRANSFUSIONS
68
studies, it is not possible to resolve the current controversy about the degree of risk concerning the association between oestrogens and endometrial carcinoma. Data from the Connecticut State Tumor Registry were supplied by Dr Lorraine Marret of the Connecticut Cancer Epidemiology Unit. R. 1. H. is a Henry J. Kaiser Family Foundation Scholar in general internal medicine.
Requests for reprints should be addressed to R. 1. H., Yale University School Medicine, Robert Wood Johnson Clinical Scholar Program, 333 Cedar Street, New Haven, Connecticut 06510, U.S.A. of
REFERENCES 1. Hutchinson GB, Rothman KJ. Correcting a bias? N Engl J Med 1978, 299: 1129-30. 2. Robboy SJ, Bradley R. Changing trends and prognostic features in endometrial cancer associated with exogenous estrogen therapy. Obstet Gynecol 1979, 54: 269-77. 3. Bauer FW, Robbins SL. An autopsy study of cancer patients JAMA 1972; 221:
1471-74 4. Suen KC, Lau LL, Yermakov V Cancer and old age. An autopsy study of 3535 patients over 65 years old. Cancer 1974; 33: 1164-68.
5. Mendenhall W, Ott L, Scheaffer RL. Elementary survey sampling. Belmont: Wadsworth, 1971. 6 Hulka BS, Grimson RC, Greenberg BG, et al. Alternative controls in a case-control study of endometrial cancer and exogenous estrogens. Am J Epidemiol 1980; 112: 376-87.
Preliminary Communications
between husband and wife could account for TLX antigens shared between the blastocyst and the mother.l5 Thus, the TLX compatible embryo might not stimulate maternal protecting or blocking factors, and the blastocyst would be rejected like any other group of foreign antigens implanted into the uterus. 16
On this evidence it should be possible to artificially stimulate protective factor production in the mother by infusing her with allogeneic leucocytes, thereby protecting the fertilised egg at implantation. We tested this hypothesis in four aborting couples with a high degree of HLA antigen sharing by infusing the women throughout pregnancy with leucocytes from many different donors. Three of these women, none of whom had passed the twelfth week in any previous pregnancy, had normal pregnancies and produced healthy babies. PATIENTS
The four couples in our study were fully informed about the potential hazards of multiple transfusions and they were
investigated to discover the cause of their recurrent abortions. No gynaecological or hormonal abnormalities were found in three of the couples. In the fourth, the wife underwent repair of a bicornuate after eight spontaneous abortions since the operation. uterus
PREVENTION OF RECURRENT ABORTION WITH LEUCOCYTE TRANSFUSIONS
abortions, but has had
two more
METHODS
COLIN TAYLOR
Three women, each with a history of three spontaneous abortions, were typed for A, B, C, and DR histocompatibility (HLA) antigens and found to share antigens with their husbands. The women were repeatedly transfused throughout pregnancy with leucocyte-enriched plasma from at least sixteen different erythrocyte-compatible donors. The pregnancies were normal and each mother produced a healthy baby. The presence of trophoblast/lymphocyte cross-reactive (TLX) antigens, which stimulate the mother to mount a response with blastocyst protective factors and which prevent maternal rejection of the antigenically unique embryo, might explain these results.
Couples were HLA typed according to the method ofMcIntyre. 17 We prepared concentrates of white cells using whole blood from two to five donors. Blood was taken into standard acid/citrate/dextrose (ACD) donor packs and tested for HBsAg by the ’Hepatest’ technique. The whole blood units were kept for at least three days in order to reduce the risk of cytomegalovirus infection. The buffy coats from each unit were removed but the lymphocyte population was not separated from the other white cells, since to do this would increase the chance of accidentally infecting the white cell concentrates. The patients were typed for ABO, Rhesus, Kell, Duffy, Lewis, Kidd, and MNSs blood groups. The erythrocyte groups of the donor units were selected in order not to stimulate red cell antibodies in the mother, and all the red cells were crossmatched against maternal sera. The concentrates were packed to reduce as much as possible the number of red cells transfused the amount of plasma proteins, and the number of platelets. The transfusions of leucocyte concentrates were repeated at intervals of about three weeks (see table). The volume of the concentrates was about 250 ml and they were infused over an hour. Patients’ pulses and blood pressures were recorded during this time. Immunopathological studies of placentae performed with anti-immunoglobulin and anti-complement sera were done according to the method of Faulk and Johnson. 18
INTRODUCTION
RESULTS
RECURRENT spontaneous abortion is associated with conditions such as deformed uterus and chromosomal abnormality, but the cause in most cases is unknown. Many investigators think that an ill-defined immunological process is responsible and there is evidence which supports this idea:
Three patients had normal pregnancies and gave birth to healthy babies (see table). The fourth patient has attained her twenty-eighth week of intrauterine pregnancy with a viable fetus according to ultrasound studies. There were no consistent immunopathological findings in any of the three placentae. None of the transfused patients has shown signs or symptoms of anti-erythrocyte antibodies, infections, anaphylactic reactions, or other complications, and the babies and their placentae were normal.
Department of Haematology, Pembury Hospital, Tunbridge Wells, Kent
W. PAGE FAULK Blond McIndoe Centre for
Transplantation Biology,
East
Grinstead,
Sussex
Summary
I.-Aborting couples share more HLA antigens of the A and B loci than would be expected by chancel-3 (an observation we have confirmed and extended to include the C and DR loci). 2.-Chronic aborters do not produce the inhibitors of cellmediated immunity4,5 normally found in maternal blood during
pregnancy.6
3.-Studies have revealed the absence of transplantation and the presence of discrete antigens 10-12 in human trophoblast membranes, which could form a basis for immune reactions between mother and fetus. 4.-Some trophoblast antigens are allotypic13 and shared with leucocytes,14 which suggests that antigens such as HLA shared
antigens7-9
DISCUSSION
suggest that multiple transfusion of useful treatment in the prevention of pooled leucocytes in three cases. The HLA abortion our spontaneous in these and other reported cases of couples compatibility From
our
results,
we
was a
69
SUMMARY OF PATIENTS AND RESULTS
who have
spontaneous abortions supports the that HLA suggestion incompatibility tends to perpetuate in human genetic diversity populations. tIt is not clear whether the success of our procedure is similar to that obtained by transfusing recipients before kidney grafting,’9 but the identification of the maternal immune responses to the transfusions may help to identify an antigen of potential use in transplantation. Shared HLA antigens between couples are not uniquely associated with spontaneous abortion. Recurrent abnormal pregnancies of unknown caused neural tube defects,21 and pre-eclamptic toxaemia are also reported associations.2z The incidence of pre-eclampsia is decreased in patients who have had a blood transfusion before their first pregnancy. 23 In addition, a woman who has had normal pregnancies can subsequently have a spontaneous abortion24 or become preeclamptic2s,26 if she becomes pregnant by another man, which raises the possibility that her abnormal pregnancy was the genetic consequence of fertilisation by a new partner and unrelated to maternal immunological defects. Human reproduction seems to be partly dependent on HLA incompatibility between husband and wife. In those women who are HLA compatible with their partners a successful pregnancy may result from a change of partner or multiple transfusions of allogeneic leucocytes before and during gestation. The point of contact between the maternal and embryonic tissues after implantation is the blastocyst, the covering of which is composed of trophoblast membranes made up of maternal and paternal gene products. HLAnegative trophoblast constitutes the materno-fetal interface in human pregnancy and it appears in these cases that TLX antigens on the transfused leucocytes triggered maternal recognition and acceptance of the embryo. We propose that these antigens, in chemically isolated form, may not only be useful in organ transplantation, but also provide a treatment for the prevention of abortion.
experienced
We thank Mr G. Hill and Mr B. Pickles ofPembury
Hospital, and Prof. Sir
John Dewhurst and NIr J. Bennett of Queen Charlotte’s Hospital, for referring patients; and Mr P. Apps and Mr R. Slater, medical laboratory scientific
officers, Pembury Hospital, and Miss Pamela Sharpe, tissue typing laboratory, Blond Mclndoe Centre for Transplantation Biology, for technical assistance. Dr J. McIntyre helped during the study. Dr M. Bayhs performed the endocrinological tests. This work was supported by the Medical Research Council, the East Gnnstead Research Trust, and the Haematology Research Fund, Pembury Hospital.
Requests for reprints should be
addressed
to
W. P. F.
REFERENCES 1. Komlos
L, Zamir R, Joshua H, Halbrecht I Common HLA antigens in couples with repeated abortions. Clin Immunol Immunopathol 1977; 7: 330-35 2. Gerencer M, Drazancic A, Kuvacic I. HLA antigen studies in women with recurrent gestational disorders Fertil Steril 1979, 31: 401-04 3 Komlos L, Halbrecht I. Repeated abortions and histocompatibility antigens. Can HLA antigen restricted gene dose effects influence the feto-maternal relationship. Med Hypotheses 1979, 5: 901-08 4. Rocklin RE, Kitzmiller J, Carpenter B, Garovoy M, David JR. Maternal-fetal relation: absence of an immunologic blocking factor from the serum of women with chronic abortions. N Engl J Med 1976; 295: 1209-13 5. Stimson WH, Strachan AF, Shepherd A Studies on the maternal immune response to placental antigens: absence of a blocking factor from the blood of abortion-prone women Br J Obstet Gynaecol 1979; 86: 41-45 6. McIntyre JA, Faulk WP. Maternal blocking factors in human pregnancy are found in plasma not serum. Lancet 1979; ii: 821-23 7. Faulk WP, Temple A. Distribution of &bgr;2-microglobulin and HLA in chorionic villi of human placentae. Nature 1976; 262: 799-802. 8. Goodfellow PH, Barnstable CJ, Bodmer WF, Snary D, Crumpton M. Expression of HLA system antigens in placenta Transplantation 1976, 22: 595-603 9. Faulk WP, Sanderson A, Temple A Distribution of MHC antigens in human placentae. Transplant Proc 1977; 9: 1379-84. 10. Faulk WP, Lovins R, Yeager C, Temple A. Antigens of human trophoblast: immunological and biochemical characterization. In: Boettcher B, ed Immunological influence on human fertility. London Academic Press, 1977: 152-60.
11. 12.
13.
Whyte A, Loke YW Antigens of the human trophoblast plasma membrane. Clin Exp Immunol 1979; 37: 359-66. Ogbimi AO, Johnson PM. Further immunological and chemical characterisation of human syncytiotrophoblast microvillous plasma membrane-associated Immunol 1980; 2: 99-108. components. Reprod J Faulk WP, McIntyre JA, Hsi Bae-Li B. Transplantation analogies of the materno-fetal relationship in human pregnancy In Touraine JL, Traeger J, Betuel H, et al, eds. Transplantation and Clinical Immunology Amsterdam. Excerpta Medica, 1979:
143-50. 14. Faulk WP, Temple A, Lovins R, Smith NC Antigens of human trophoblast: a working hypothesis for their role in normal and abnormal pregnancies Proc Natl Acad Sci U.S.A 1978; 75: 1947-51. 15. Faulk WP, McIntyre JA. Trophoblast survival. Transplantation (in press). 16. Beer AE, Billingham RE Host responses to intra-uterine tissue, cellular and fetal allografts. J Reprod Fert 974; suppl 21: 59-88.
References continued overleaf
70
0 -35%)
was pumped through at a superficial velocity of 1 -9x 10’m/s. This corresponded to a flow rate of 3 ml/min. This was followed by phosphate-buffered saline for about 10 minutes, the effluent from the chamber being by that time almost colourless. The
Methods and Devices
filter was removed from the magnet and the flow rate increased to 10 ml/min to flush out any cells that had been captured. Blood films were made from the original suspension, the effluent from the column, and the suspension flushed from the filter. Thin films were prepared, fixed in methanol, and stained with Giemsa stain. Percentages are based on counts of 10 000-60 000 erythrocytes. In a second series of experiments a small perspex chamber 1 - 3 cm x 1 - 3 cm x 0 -9cm was packed with the same steel wire to a packing density of 7 -6%. In this case 2 ml of blood with 5% haematocrit and a lower initial parasite concentration (0-035%) was used. The applied magnetic field was as before, but various flow rates were used, 10 ml of fluid being collected in the effluent and flush cycles.
SEPARATION OF MALARIA-INFECTED ERYTHROCYTES FROM WHOLE BLOOD: USE OF A SELECTIVE HIGH-GRADIENT MAGNETIC SEPARATION TECHNIQUE F. PAUL
S. ROATH D. MELVILLE
Departments of Haematology and Physics, University of Southampton D. C. WARHURST
J. O. S. OSISANYA
Amoebiasis Unit, Hospital for Tropical Diseases, and Department Medical Protozoology, London School of Hygiene and Tropical Medicine
of Results In the first were
MALARIA can be difficult to diagnose because of the small numbers of parasitised cells in an infected person’s blood. Low concentrations of malarial parasites also hamper research into, for example, vaccine production. Density-gradient or osmotic-fragility techniques are normally used to obtain increased concentrations of parasites, but these have had only limited success. It is known, however, that the malarial parasite digests haemoglobin to leave high-spin oxidised haem products. These are paramagnetic, in contrast to normal low-spin oxyhaemoglobin, which is diamagnetic. Consequently it should be possible to separate parasitised erythrocytes from normal oxygenated ones with a high-gradient magnetic-separation technique which will selectively separate such a high-spin form of haemoglobin (and the cells containing it). This technique has already been shown to separate deoxygenated erythrocytes from blood because of their similar qualities (high spin and paramagnetism).!
experiment
75% of the
trophozoites
and schizonts
extracted, and these had been concentrated 45-fold. The
processing time was approximately
15 min
(see table).
CONCENTRATION OF TROPHOZOITES AND SCHIZONTS ACHIEVED WITH HIGH-GRADIENT MAGNETIC SEPARATION
’
Methods
The results of one of the second series of experiments carried out a flow rate corresponding to a superficial velocity of 1 · 8 x 10- 4m/s or 1-3ml/min are also shown in the table. In this case a 42-fold concentration was achieved for schizonts and trophozoites in a processing time of about 8 min. The number of parasitised red cells captured on the filter was approximately 107 in the first experiment and 105 in the second. From previous datasuch low values indicate that throughout the experiments the wire can be regarded as "clea" and that much higher capture levels and hence concentration ratios can be achieved. at
A culture of Plasmodium falciparum was used throughout. For the first experiment a ’Perspex’ chamber 2 - 5 cm x 1 cm x 5 - 2 cm was filled to a packing density of 4’ 6% with 25 j.tm diameter 430-grade stainless-steel wire and placed between the poles of a permanent magnet which generated a uniform field of 0 -77 tesla. The chamber was flushed with phosphate-buffered saline, and then approximately 10 ml of the plasmodium culture containing 10% by volume erythrocytes (initial parasitised-cell concentration
Discussion 17.
McIntyre JA Laboratory techniques. In Munster AW, ed. Surgical immunology
New
York: Grune &
Stratton, 1976: 283-306. Johnson PM. Immunological studies of
human placentae identification and distribution of proteins in mature chorionic villi. Clin Exp Immunol 1977, 27: 365-75. 19. Williams IA, Ting A, French ME, Oliver D, Morris PJ. Preoperative bloodtransfusions improve cadaveric renal allograft survival in non-transfused recipients Lancet 1980; i: 1104-06. 20. Gerencer M, Kastelan A, Drazancic A, Kerhin-Brkljacic V, Madjaric M. The HLA antigens in women with recurrent abnormal pregnancies of unknown etiology. Tissue Antigens 1978; 12: 223-27 21 Schacter B, Muir A, Gyves M, Tasin M HLA-A,B compatibility in parents of offspring with neural-tube defects or couples experiencing involuntary fetal wastage. Lancet 1979; i. 796-99. 22. Redman CWG. Immunological aspects of eclampsia and pre-eclampsia. In: Hearn JP, ed. Immunological Aspects of Reproduction and Fertility Control Lancaster- MTP Press, 1980: 83-103. 23. Feeney JG, Tovey LAD, Scott JS. Influence of previous blood-transfusion on incidence of pre-eclampsia. Lancet 1977; i: 874-75. 24. Javert CT. Further follow-up on habitual abortion patients. Am J Obstet Gynecol 1962; 84: 1149-59. 25. Need JA. Pre-eclampsia in pregnancy by different fathers: Immunological studies. Br Med J 1975, 1: 548-49 26. Feeney JG Pre-eclampsia and changed paternity In: Bonnar J, Macgillivray I, Symonds M, eds. Pregnancy hypertension. Lancaster: MTP Press, 1980 41-44. 18. Faulk WP,
An attempt to use the magnetic properties of blood cells containing malarial parasites to effect separation was reported by Heidelberger3in 1946. The low field gradient available at that time resulted in processing times of the order of 6-12 h. We use a highgradient magnetic separation technique, in which the very high magnetic-field gradients surrounding fine ferromagnetic wires in a uniform magnetic field are used to translocate small particles. The field gradients available are about x 1000 those available to Heidelberger, and consequently our processing times are shorter. All our experiments show a clear enhancement of schizonts and trophozoites at initial parasitaemias of 0-35% and 0-035%. These low levels were chosen as being similar to levels found in malaria patients and indicate that the method can be used on fresh blood
without further incubation. tests on the parasitised red cells have previously reported extensive studies of both morphology and viability for red cells, white cells, and platelets which have been magnetically filtered,4and we found no detectable damage which can be attributed to the process for, these blood components.
We did not carry out
viability
separated by the filter, but
we
studies, it is not possible to resolve the current controversy about the degree of risk concerning the association between oestrogens and endometrial carcinoma. Data from the Connecticut State Tumor Registry were supplied by Dr Lorraine Marret of the Connecticut Cancer Epidemiology Unit. R. 1. H. is a Henry J. Kaiser Family Foundation Scholar in general internal medicine.
Requests for reprints should be addressed to R. 1. H., Yale University School Medicine, Robert Wood Johnson Clinical Scholar Program, 333 Cedar Street, New Haven, Connecticut 06510, U.S.A. of
REFERENCES 1. Hutchinson GB, Rothman KJ. Correcting a bias? N Engl J Med 1978, 299: 1129-30. 2. Robboy SJ, Bradley R. Changing trends and prognostic features in endometrial cancer associated with exogenous estrogen therapy. Obstet Gynecol 1979, 54: 269-77. 3. Bauer FW, Robbins SL. An autopsy study of cancer patients JAMA 1972; 221:
1471-74 4. Suen KC, Lau LL, Yermakov V Cancer and old age. An autopsy study of 3535 patients over 65 years old. Cancer 1974; 33: 1164-68.
5. Mendenhall W, Ott L, Scheaffer RL. Elementary survey sampling. Belmont: Wadsworth, 1971. 6 Hulka BS, Grimson RC, Greenberg BG, et al. Alternative controls in a case-control study of endometrial cancer and exogenous estrogens. Am J Epidemiol 1980; 112: 376-87.
Preliminary Communications
between husband and wife could account for TLX antigens shared between the blastocyst and the mother.l5 Thus, the TLX compatible embryo might not stimulate maternal protecting or blocking factors, and the blastocyst would be rejected like any other group of foreign antigens implanted into the uterus. 16
On this evidence it should be possible to artificially stimulate protective factor production in the mother by infusing her with allogeneic leucocytes, thereby protecting the fertilised egg at implantation. We tested this hypothesis in four aborting couples with a high degree of HLA antigen sharing by infusing the women throughout pregnancy with leucocytes from many different donors. Three of these women, none of whom had passed the twelfth week in any previous pregnancy, had normal pregnancies and produced healthy babies. PATIENTS
The four couples in our study were fully informed about the potential hazards of multiple transfusions and they were
investigated to discover the cause of their recurrent abortions. No gynaecological or hormonal abnormalities were found in three of the couples. In the fourth, the wife underwent repair of a bicornuate after eight spontaneous abortions since the operation. uterus
PREVENTION OF RECURRENT ABORTION WITH LEUCOCYTE TRANSFUSIONS
abortions, but has had
two more
METHODS
COLIN TAYLOR
Three women, each with a history of three spontaneous abortions, were typed for A, B, C, and DR histocompatibility (HLA) antigens and found to share antigens with their husbands. The women were repeatedly transfused throughout pregnancy with leucocyte-enriched plasma from at least sixteen different erythrocyte-compatible donors. The pregnancies were normal and each mother produced a healthy baby. The presence of trophoblast/lymphocyte cross-reactive (TLX) antigens, which stimulate the mother to mount a response with blastocyst protective factors and which prevent maternal rejection of the antigenically unique embryo, might explain these results.
Couples were HLA typed according to the method ofMcIntyre. 17 We prepared concentrates of white cells using whole blood from two to five donors. Blood was taken into standard acid/citrate/dextrose (ACD) donor packs and tested for HBsAg by the ’Hepatest’ technique. The whole blood units were kept for at least three days in order to reduce the risk of cytomegalovirus infection. The buffy coats from each unit were removed but the lymphocyte population was not separated from the other white cells, since to do this would increase the chance of accidentally infecting the white cell concentrates. The patients were typed for ABO, Rhesus, Kell, Duffy, Lewis, Kidd, and MNSs blood groups. The erythrocyte groups of the donor units were selected in order not to stimulate red cell antibodies in the mother, and all the red cells were crossmatched against maternal sera. The concentrates were packed to reduce as much as possible the number of red cells transfused the amount of plasma proteins, and the number of platelets. The transfusions of leucocyte concentrates were repeated at intervals of about three weeks (see table). The volume of the concentrates was about 250 ml and they were infused over an hour. Patients’ pulses and blood pressures were recorded during this time. Immunopathological studies of placentae performed with anti-immunoglobulin and anti-complement sera were done according to the method of Faulk and Johnson. 18
INTRODUCTION
RESULTS
RECURRENT spontaneous abortion is associated with conditions such as deformed uterus and chromosomal abnormality, but the cause in most cases is unknown. Many investigators think that an ill-defined immunological process is responsible and there is evidence which supports this idea:
Three patients had normal pregnancies and gave birth to healthy babies (see table). The fourth patient has attained her twenty-eighth week of intrauterine pregnancy with a viable fetus according to ultrasound studies. There were no consistent immunopathological findings in any of the three placentae. None of the transfused patients has shown signs or symptoms of anti-erythrocyte antibodies, infections, anaphylactic reactions, or other complications, and the babies and their placentae were normal.
Department of Haematology, Pembury Hospital, Tunbridge Wells, Kent
W. PAGE FAULK Blond McIndoe Centre for
Transplantation Biology,
East
Grinstead,
Sussex
Summary
I.-Aborting couples share more HLA antigens of the A and B loci than would be expected by chancel-3 (an observation we have confirmed and extended to include the C and DR loci). 2.-Chronic aborters do not produce the inhibitors of cellmediated immunity4,5 normally found in maternal blood during
pregnancy.6
3.-Studies have revealed the absence of transplantation and the presence of discrete antigens 10-12 in human trophoblast membranes, which could form a basis for immune reactions between mother and fetus. 4.-Some trophoblast antigens are allotypic13 and shared with leucocytes,14 which suggests that antigens such as HLA shared
antigens7-9
DISCUSSION
suggest that multiple transfusion of useful treatment in the prevention of pooled leucocytes in three cases. The HLA abortion our spontaneous in these and other reported cases of couples compatibility From
our
results,
we
was a
69
SUMMARY OF PATIENTS AND RESULTS
who have
spontaneous abortions supports the that HLA suggestion incompatibility tends to perpetuate in human genetic diversity populations. tIt is not clear whether the success of our procedure is similar to that obtained by transfusing recipients before kidney grafting,’9 but the identification of the maternal immune responses to the transfusions may help to identify an antigen of potential use in transplantation. Shared HLA antigens between couples are not uniquely associated with spontaneous abortion. Recurrent abnormal pregnancies of unknown caused neural tube defects,21 and pre-eclamptic toxaemia are also reported associations.2z The incidence of pre-eclampsia is decreased in patients who have had a blood transfusion before their first pregnancy. 23 In addition, a woman who has had normal pregnancies can subsequently have a spontaneous abortion24 or become preeclamptic2s,26 if she becomes pregnant by another man, which raises the possibility that her abnormal pregnancy was the genetic consequence of fertilisation by a new partner and unrelated to maternal immunological defects. Human reproduction seems to be partly dependent on HLA incompatibility between husband and wife. In those women who are HLA compatible with their partners a successful pregnancy may result from a change of partner or multiple transfusions of allogeneic leucocytes before and during gestation. The point of contact between the maternal and embryonic tissues after implantation is the blastocyst, the covering of which is composed of trophoblast membranes made up of maternal and paternal gene products. HLAnegative trophoblast constitutes the materno-fetal interface in human pregnancy and it appears in these cases that TLX antigens on the transfused leucocytes triggered maternal recognition and acceptance of the embryo. We propose that these antigens, in chemically isolated form, may not only be useful in organ transplantation, but also provide a treatment for the prevention of abortion.
experienced
We thank Mr G. Hill and Mr B. Pickles ofPembury
Hospital, and Prof. Sir
John Dewhurst and NIr J. Bennett of Queen Charlotte’s Hospital, for referring patients; and Mr P. Apps and Mr R. Slater, medical laboratory scientific
officers, Pembury Hospital, and Miss Pamela Sharpe, tissue typing laboratory, Blond Mclndoe Centre for Transplantation Biology, for technical assistance. Dr J. McIntyre helped during the study. Dr M. Bayhs performed the endocrinological tests. This work was supported by the Medical Research Council, the East Gnnstead Research Trust, and the Haematology Research Fund, Pembury Hospital.
Requests for reprints should be
addressed
to
W. P. F.
REFERENCES 1. Komlos
L, Zamir R, Joshua H, Halbrecht I Common HLA antigens in couples with repeated abortions. Clin Immunol Immunopathol 1977; 7: 330-35 2. Gerencer M, Drazancic A, Kuvacic I. HLA antigen studies in women with recurrent gestational disorders Fertil Steril 1979, 31: 401-04 3 Komlos L, Halbrecht I. Repeated abortions and histocompatibility antigens. Can HLA antigen restricted gene dose effects influence the feto-maternal relationship. Med Hypotheses 1979, 5: 901-08 4. Rocklin RE, Kitzmiller J, Carpenter B, Garovoy M, David JR. Maternal-fetal relation: absence of an immunologic blocking factor from the serum of women with chronic abortions. N Engl J Med 1976; 295: 1209-13 5. Stimson WH, Strachan AF, Shepherd A Studies on the maternal immune response to placental antigens: absence of a blocking factor from the blood of abortion-prone women Br J Obstet Gynaecol 1979; 86: 41-45 6. McIntyre JA, Faulk WP. Maternal blocking factors in human pregnancy are found in plasma not serum. Lancet 1979; ii: 821-23 7. Faulk WP, Temple A. Distribution of &bgr;2-microglobulin and HLA in chorionic villi of human placentae. Nature 1976; 262: 799-802. 8. Goodfellow PH, Barnstable CJ, Bodmer WF, Snary D, Crumpton M. Expression of HLA system antigens in placenta Transplantation 1976, 22: 595-603 9. Faulk WP, Sanderson A, Temple A Distribution of MHC antigens in human placentae. Transplant Proc 1977; 9: 1379-84. 10. Faulk WP, Lovins R, Yeager C, Temple A. Antigens of human trophoblast: immunological and biochemical characterization. In: Boettcher B, ed Immunological influence on human fertility. London Academic Press, 1977: 152-60.
11. 12.
13.
Whyte A, Loke YW Antigens of the human trophoblast plasma membrane. Clin Exp Immunol 1979; 37: 359-66. Ogbimi AO, Johnson PM. Further immunological and chemical characterisation of human syncytiotrophoblast microvillous plasma membrane-associated Immunol 1980; 2: 99-108. components. Reprod J Faulk WP, McIntyre JA, Hsi Bae-Li B. Transplantation analogies of the materno-fetal relationship in human pregnancy In Touraine JL, Traeger J, Betuel H, et al, eds. Transplantation and Clinical Immunology Amsterdam. Excerpta Medica, 1979:
143-50. 14. Faulk WP, Temple A, Lovins R, Smith NC Antigens of human trophoblast: a working hypothesis for their role in normal and abnormal pregnancies Proc Natl Acad Sci U.S.A 1978; 75: 1947-51. 15. Faulk WP, McIntyre JA. Trophoblast survival. Transplantation (in press). 16. Beer AE, Billingham RE Host responses to intra-uterine tissue, cellular and fetal allografts. J Reprod Fert 974; suppl 21: 59-88.
References continued overleaf
70
0 -35%)
was pumped through at a superficial velocity of 1 -9x 10’m/s. This corresponded to a flow rate of 3 ml/min. This was followed by phosphate-buffered saline for about 10 minutes, the effluent from the chamber being by that time almost colourless. The
Methods and Devices
filter was removed from the magnet and the flow rate increased to 10 ml/min to flush out any cells that had been captured. Blood films were made from the original suspension, the effluent from the column, and the suspension flushed from the filter. Thin films were prepared, fixed in methanol, and stained with Giemsa stain. Percentages are based on counts of 10 000-60 000 erythrocytes. In a second series of experiments a small perspex chamber 1 - 3 cm x 1 - 3 cm x 0 -9cm was packed with the same steel wire to a packing density of 7 -6%. In this case 2 ml of blood with 5% haematocrit and a lower initial parasite concentration (0-035%) was used. The applied magnetic field was as before, but various flow rates were used, 10 ml of fluid being collected in the effluent and flush cycles.
SEPARATION OF MALARIA-INFECTED ERYTHROCYTES FROM WHOLE BLOOD: USE OF A SELECTIVE HIGH-GRADIENT MAGNETIC SEPARATION TECHNIQUE F. PAUL
S. ROATH D. MELVILLE
Departments of Haematology and Physics, University of Southampton D. C. WARHURST
J. O. S. OSISANYA
Amoebiasis Unit, Hospital for Tropical Diseases, and Department Medical Protozoology, London School of Hygiene and Tropical Medicine
of Results In the first were
MALARIA can be difficult to diagnose because of the small numbers of parasitised cells in an infected person’s blood. Low concentrations of malarial parasites also hamper research into, for example, vaccine production. Density-gradient or osmotic-fragility techniques are normally used to obtain increased concentrations of parasites, but these have had only limited success. It is known, however, that the malarial parasite digests haemoglobin to leave high-spin oxidised haem products. These are paramagnetic, in contrast to normal low-spin oxyhaemoglobin, which is diamagnetic. Consequently it should be possible to separate parasitised erythrocytes from normal oxygenated ones with a high-gradient magnetic-separation technique which will selectively separate such a high-spin form of haemoglobin (and the cells containing it). This technique has already been shown to separate deoxygenated erythrocytes from blood because of their similar qualities (high spin and paramagnetism).!
experiment
75% of the
trophozoites
and schizonts
extracted, and these had been concentrated 45-fold. The
processing time was approximately
15 min
(see table).
CONCENTRATION OF TROPHOZOITES AND SCHIZONTS ACHIEVED WITH HIGH-GRADIENT MAGNETIC SEPARATION
’
Methods
The results of one of the second series of experiments carried out a flow rate corresponding to a superficial velocity of 1 · 8 x 10- 4m/s or 1-3ml/min are also shown in the table. In this case a 42-fold concentration was achieved for schizonts and trophozoites in a processing time of about 8 min. The number of parasitised red cells captured on the filter was approximately 107 in the first experiment and 105 in the second. From previous datasuch low values indicate that throughout the experiments the wire can be regarded as "clea" and that much higher capture levels and hence concentration ratios can be achieved. at
A culture of Plasmodium falciparum was used throughout. For the first experiment a ’Perspex’ chamber 2 - 5 cm x 1 cm x 5 - 2 cm was filled to a packing density of 4’ 6% with 25 j.tm diameter 430-grade stainless-steel wire and placed between the poles of a permanent magnet which generated a uniform field of 0 -77 tesla. The chamber was flushed with phosphate-buffered saline, and then approximately 10 ml of the plasmodium culture containing 10% by volume erythrocytes (initial parasitised-cell concentration
Discussion 17.
McIntyre JA Laboratory techniques. In Munster AW, ed. Surgical immunology
New
York: Grune &
Stratton, 1976: 283-306. Johnson PM. Immunological studies of
human placentae identification and distribution of proteins in mature chorionic villi. Clin Exp Immunol 1977, 27: 365-75. 19. Williams IA, Ting A, French ME, Oliver D, Morris PJ. Preoperative bloodtransfusions improve cadaveric renal allograft survival in non-transfused recipients Lancet 1980; i: 1104-06. 20. Gerencer M, Kastelan A, Drazancic A, Kerhin-Brkljacic V, Madjaric M. The HLA antigens in women with recurrent abnormal pregnancies of unknown etiology. Tissue Antigens 1978; 12: 223-27 21 Schacter B, Muir A, Gyves M, Tasin M HLA-A,B compatibility in parents of offspring with neural-tube defects or couples experiencing involuntary fetal wastage. Lancet 1979; i. 796-99. 22. Redman CWG. Immunological aspects of eclampsia and pre-eclampsia. In: Hearn JP, ed. Immunological Aspects of Reproduction and Fertility Control Lancaster- MTP Press, 1980: 83-103. 23. Feeney JG, Tovey LAD, Scott JS. Influence of previous blood-transfusion on incidence of pre-eclampsia. Lancet 1977; i: 874-75. 24. Javert CT. Further follow-up on habitual abortion patients. Am J Obstet Gynecol 1962; 84: 1149-59. 25. Need JA. Pre-eclampsia in pregnancy by different fathers: Immunological studies. Br Med J 1975, 1: 548-49 26. Feeney JG Pre-eclampsia and changed paternity In: Bonnar J, Macgillivray I, Symonds M, eds. Pregnancy hypertension. Lancaster: MTP Press, 1980 41-44. 18. Faulk WP,
An attempt to use the magnetic properties of blood cells containing malarial parasites to effect separation was reported by Heidelberger3in 1946. The low field gradient available at that time resulted in processing times of the order of 6-12 h. We use a highgradient magnetic separation technique, in which the very high magnetic-field gradients surrounding fine ferromagnetic wires in a uniform magnetic field are used to translocate small particles. The field gradients available are about x 1000 those available to Heidelberger, and consequently our processing times are shorter. All our experiments show a clear enhancement of schizonts and trophozoites at initial parasitaemias of 0-35% and 0-035%. These low levels were chosen as being similar to levels found in malaria patients and indicate that the method can be used on fresh blood
without further incubation. tests on the parasitised red cells have previously reported extensive studies of both morphology and viability for red cells, white cells, and platelets which have been magnetically filtered,4and we found no detectable damage which can be attributed to the process for, these blood components.
We did not carry out
viability
separated by the filter, but
we