- Email: [email protected]
Transplacental passage of maternal erythrocytes with sickling
November 1975
The Journal o f P E D I A T R I C S
781
Transplacental passage of maternal erythrocytes with sickling Siekled erythrocytes served as a marker for maternal blood in a cell transfer study o f placentas from uncomplicated term pregnancies of women with sicklemia. The incidence of concurrent sickling in maternal and fetal blood was 100% in the 44 cases examined. Maternofetal passage of the erythrocytes was found four times more frequently in umbilical or chorionic veins (8.4%) than in arteries (1.9%). Deported ehorionic villi were found in the veins in three of 44 eases but not in the arteries. These findings indicate fresh maternofetal leakage of blood during the third stage of labor.
Toshio Fujikura, M.D.,* and Bernard Klionsky, M.D., Pittsburgh, Pa.
THE PRESENCE of fetal erythrocytes in the maternal circulation has been amply documented. The passage of erythrocytes and leukocytes from fetus to mother occurs mainly at the time of delivery. The literature with respect to the passage of maternal cells into the fetal circulation, on the other hand, is conflicting. Human studies of maternofetal passage have involved the administration of blood transfusions to mothers with isotope-labeled erythrocytes, 1 sickled erythrocytes, ~ or elliptocytes:~ prior to delivery. These human experiments demonstrate that injected erythrocytes traverse the placental barrier and are found in appreciable numbers in the fetal circulation. An immunofluorescent-labeled study ~ has indicated, however, that passage of maternal cells into the fetus is an exceptional occurrence of minimal quantitative significance, and that the reported presence of such cells in the fetal circulation may reflect contamination of cord blood. With the usual histopathologic methods of tissue processing and formalin fixation, the sickling state is mainFrom the University of Pittsburgh, School of Medicine, Department of Pathology. The Collaborative Study of Cerebral Palsy, Mental Retardation, and Other Neurological and Sensory Disorders of lnfancy and Childhood is supported by the National Institute of Neurological Diseases and Stroke, National Institute of Health. *Reprint address: Magee-Womens Hospital, Department of Pathology, ForbesA venue and Halket St., Pittsburgh, Pa. 15213.
tained in the delivered placenta and umbilical cord, providing an excellent tool for the diagnosis of sicklemia? Since the sickling rate of fetal erythrocytes has been reported to be extremely low at birth? -~ transplacental passage of maternal erythrocytes into the fetal circulation can be estimated by using the sickling phenomenon as a tracer. The maternal-fetal leakage of blood may exert a significant influence on the immunologic reactivity .of the infant. This is particularly important in the case of intrauterine exposure of the female Rh-negative fetus to Rh antigens from an Rh-positive mother. The purpose of this study is to demonstrate quantitatively transplacental passage of sickled maternal erythrocytes into the fetal circulation and to indicate that the maternofetal passage is as common as the fetomaternal passage of blood.
MATERIAL AND METHODS The placentas of patients known to have sicklemia were selected from Collaborative Study materials obtained from the University of Tennessee, Medical School Hospital, Memphis, Tennessee. The majority of the patients had sickle cell trait but hemoglobin electrophoreses were not routinely done. Only single live births, delivered spontaneously beyond 37 weeks of gestation and without maternal complications, were included in the study. For the proper calculation of sickling, only those showing a sickling rate of more than 90% in the intervillous space of the cotyledonary lobule were included.
Vol. 87, No. 5, pp. 781,783
782
Fujikura and Klionsky
The Journal of Pediatrics November 1975
4.1%. Small numbers of sickled erythrocytes were found in the fetal circulation of the placenta, especially in the umbilical veins. The incidence of concurrent sickling in maternal a n d fetal blood was 100% in the 44 cases selected. The mean sickling rate in umbilical or chorionic veins (8.4 _+ 5.1%) of 27 cases was higher than in those arteries (1.9 _+ 1.1%) of 25 cases. Furthermore, three out of 44 cases showed deported chorionic villi in umbilical or chorionic veins but not in those arteries. COMMENT
Fig. 1. Sickled erythrocytes and deported villi inlarge chorionic vein (x 225). This finding indicates fresh and mass contamination of maternal blood in the fetal circulation.
Table 1. Sickling rate of erythrocytes in the fetal and maternal circulation In tervillous space cotyledonary lobule
No.-of Cases examined Mean rate (%) SD
44 95.2 4.1
Umbilical or chorionic Vein
27 8.4 5.1
Arterv
25 1.9 1.1
Ten percent formalin was the best fixative for this purpose and the usual histopathologic methods of tissue processing preserved the sickled state of maternal erythrocytes. ~ The hematoxylin-eosin sections examined were from 3 cm wide blocks spanning the entire thickness of the placenta and from a cross section of the umbilical cord. If fetal erythrocytes were intact without artefacts, erythrocytes of umbilical or chorionic veins and arteries were evaluated for sickling counts. The m e a n sickling rate was calculated by counting the number of sickled erythrocytes out of a total of 500 erythrocytes in each area. In the 44 placentas the number of cases examined for sickling counts was limited to 25 of the artery and 27 of the vein due to various artefacts. RESULTS Table I shows the mean percentage a n d standard deviation of the sickling rate in three different areas: intervillous space, umbilical vein, and arteries. Since only cases showing a sickling rate of more than 90% were included in this study, the mean sickling rate in the intervillous space of the cotyledonary lobule was 95.2% _+
Third stage leakage. Maternofetal passage of the erythrocytes was found four times more frequently in umbilical or chorionic veins than in arteries. The higher percentage of sickled red blood cells in the veins emphasizes the acute nature of the maternofetal transfusion during the third stage of labor. Umbilical veins contain oxygenated blood (mean 31 mm Hg) returning from the placenta, whereas deoxygenated blood (mean 18 mm Hg) flows from fetus in the arteriesY In vitro analysis an oxygen tension of 45 mm Hg is taken to be the threshold for sickling of erythrocytes in patients with sickle cell anemia; the threshold for sickling in those with sickle cell trait is estimated to be 18 mm Hg. TM 11 If maternofetal passage of theerythrocytes occurs before the third stage of labor, t h e h i g h e r percentage of sickled cells could be found in umbilical or chorionic arteries where oxygen tensions are lower. The presence of deported chorionic villi in umbilical or chorionic veins indicates the possibility of a large shunt between the two circulations. When the placenta has separated, the common practice of placental expression may traumatize delicate chorionic villi anchoring the decidua. If the uterus is vigorously massaged or squeezed, such an attempt may increase the chance of villous damage. Sickling phenomenon. The mean sickling rate was 95.2% in the maternal circulation and 8.4% in umbilical or chorionic veinsl However, such a high sickling rate in vitro may not accurately reflect that of the in vivo phenomenon. It is unlikely that the high sickling rate actually occurs in vivo and may interfere with the placental circulation. If the sluggish flow of maternal erythrocytes does occur through the placenta, villous thrombosis and infarction would be increased, but these problems were not commonly found in the present study. Th e placental findings with sicklemia were not significantly different from those without sicklemia. The acidity of formalin fixative produces a right shift in the oxygen dissociation curve which exerts a favorable influence on the sickling phenomenon. 12 When the placenta separates from the uterine wall, the resulting
Volume 87 Number 5 hypoxia may create a natural sickling preparation of the trapped erythrocytes in the placenta. However, oxygenated erythrocytes in the decidual arteries of these 44 placentas were free from sickling, and deoxygenated erythrocytes only showed sickling in the maternal circulation. This indicates that the fixation and time intervals after delivery are not a strong influencing factor on the sickling p h e n o m e n o n of oxygenated erythrocytes in the n a t u r a l sickling preparation of the placenta. The presence of sickling based on sickling preparations has been reported in n e w b o r n infants. 6-8 However, these reports did not consider the possibility of the maternofetal passage and no appreciable quantity of S hemoglobin was detected by paper electrophoresis. The mechanism of sickling is obscure in newborn infants, since n e w b o r n blood contains about 70-80% of the fetal hemoglobin which is unable to produce sickling. The concentration of fetal hemoglobin in patients with sickle cell a n e m i a and sickle cell trait is known to be slightly high or in the normal range. 13 Placental transfusion. The blood volume of n e w b o r n infants varies over a wide range depending on the a m o u n t of placental transfusion at birth. Placental transfusion has been shown by an increase in weight of the infant of 55 to 180 gm during the first minutes of life when the umbilical cord is left unclamped. TM The volume of the placental transfusion has been estimated to be more than 40% above the initial infant blood 7olume when the umbilical cord is clamped immediately. TM Considering the large amount of placental transfusion during the third stage of labor, delayed ligation of the cord may increase the transplacental passage of maternal antigens into the fetal circulation and predispose the fetus to the development of immunologically mediated disease upon s u b s e q u e n t exposure to the antigen. 1~ D u n n and associates TM reported that the frequency of microtransfusions from fetus to mother occurring during the third stage of labor is not influenced by the timing of cord ligation. However, similar information on maternofetal transfusion is less well known in relation to the proper timing of cord ligation. This is particularly important in premature infants for providing an adequate blood volume without maternal blood contamination. Rh isoimmunization. Rh-positive mothers may induce immunologic tolerance or primary sensitization in their Rh-negative daughters as the result of transplacental passage of the maternal erythrocytes to their daughters in utero. However, this hypothesis of immunologic tolerance has not been supported b y factual evidence2 . . . . The effect of the grandmother's blood types on the incidence of erythroblastosis among their grandchildren was studied
Transplacental passage of maternal erythrocytes
7 83
by Taylor. 18 Of the children who had Rh-positive grandmothers, 62% were affected, whereas 46% of those with Rh-negative grandmothers were affected, an increase of 22%. 11 Thus there was a highly' statistically significant relation between the grandmother's Rh type and an increased incidence of erythroblastosis in the third generation. It is interesting to investigate whether Rh-negative female infants born of Rh-positive mothers should routinely be given prophylactic anti-D i m m u n e globulin at birth.~7 REFERENCES
1. Mengert WF, Rights CS, Bates CR, Reid AF, Wolf GR, and Nabors GC: Placental transmission of erythrocytes, Am J Obstet Gynecol, 69:678, 1955. 2. Macris NT, Hellman LM, and Watson RJ: The transmission of transfused sickle-trait cells from mother to fetus, Am J Obstet Gynecol 76:1214, 1958. 3. Hedenstedt S, and Naeslund J: Investigations of the permeability of the placenta with help of elliptocytes, Acta Med Scand [Suppl] 170:126, 1946. 4. Cohen F, and Zuelzer WW: The transplacental passage of maternal erythrocytes into the fetus, Obstet Gyneco193:566, 1965. 5. Fujikura T, and Froehlich L: Diagnosis of sickling by placental examination, Am J Obstet Gynecol 100:1122, 1968. 6. Porter FS, and Thurman WF: Studies of sickle cell disease, diagnosis in infancy, Am J Dis Child 106:35, 1963. 7. Schneider RG, and Haggard MD: Sickling, a quantitatively delayed genetic character, Proc Soc Exp Biol Med 89:196, 1955. 8. Watson J: The significance of the paucity of sickle cell cells in newborn Negro infants, Am J Med Sci 215:419, 1948. 9. Marquis L, and Ackerman BD: Placental respiration in the immediate neonatal period, Am J Obstet Gynecol 117:358, 1973. 10. Allison AC: Observations on the sickling phenomenon and on the distrbution of different hemoglobin types in erythrocyte populations, Clin Sci 15:497, 1950. 11. Sherman IJ: The sickling phenomenon with special reference to the differentiation of sickle celt anemia from the sickle cell trait, Bull Johns Hopkins Hosp 67:309, 1940. 12. Beutler E: Hypothesis: Changes in the oxygen dissociation curve and sickling: A general formulation and therapeutic strategy, Blood 43:297, 1974. 13. Ross Conference on Pediatric Research: Fetal hemoglobin, Columbus, Ohio, 1964, Ross Laboratories, pp 19 and 57. 14. Yao A, and Lind J: Placental transfusion, Am J Dis Child 127:128, 1974. 15. Scott JS: Immunological diseases and pregnancy, Br Med J 1;1559, 1966. 16. Dunn PM, Frazer ID, and Raper A: Influence of early cord ligation on the traneplacental passage of feotal cells, J Obstet Gynecol Br Commonw 73:757, 1966. 17. Scott. JR: Immunological factors in first pregnancy Rh isoimmunization, Lancet 1:717, 1973. 18. Taylor JF: Sensitization of Rh-negative daughters by their Rh-positive mothers, N Engl J Med 276:547, 1967.
The Journal o f P E D I A T R I C S
781
Transplacental passage of maternal erythrocytes with sickling Siekled erythrocytes served as a marker for maternal blood in a cell transfer study o f placentas from uncomplicated term pregnancies of women with sicklemia. The incidence of concurrent sickling in maternal and fetal blood was 100% in the 44 cases examined. Maternofetal passage of the erythrocytes was found four times more frequently in umbilical or chorionic veins (8.4%) than in arteries (1.9%). Deported ehorionic villi were found in the veins in three of 44 eases but not in the arteries. These findings indicate fresh maternofetal leakage of blood during the third stage of labor.
Toshio Fujikura, M.D.,* and Bernard Klionsky, M.D., Pittsburgh, Pa.
THE PRESENCE of fetal erythrocytes in the maternal circulation has been amply documented. The passage of erythrocytes and leukocytes from fetus to mother occurs mainly at the time of delivery. The literature with respect to the passage of maternal cells into the fetal circulation, on the other hand, is conflicting. Human studies of maternofetal passage have involved the administration of blood transfusions to mothers with isotope-labeled erythrocytes, 1 sickled erythrocytes, ~ or elliptocytes:~ prior to delivery. These human experiments demonstrate that injected erythrocytes traverse the placental barrier and are found in appreciable numbers in the fetal circulation. An immunofluorescent-labeled study ~ has indicated, however, that passage of maternal cells into the fetus is an exceptional occurrence of minimal quantitative significance, and that the reported presence of such cells in the fetal circulation may reflect contamination of cord blood. With the usual histopathologic methods of tissue processing and formalin fixation, the sickling state is mainFrom the University of Pittsburgh, School of Medicine, Department of Pathology. The Collaborative Study of Cerebral Palsy, Mental Retardation, and Other Neurological and Sensory Disorders of lnfancy and Childhood is supported by the National Institute of Neurological Diseases and Stroke, National Institute of Health. *Reprint address: Magee-Womens Hospital, Department of Pathology, ForbesA venue and Halket St., Pittsburgh, Pa. 15213.
tained in the delivered placenta and umbilical cord, providing an excellent tool for the diagnosis of sicklemia? Since the sickling rate of fetal erythrocytes has been reported to be extremely low at birth? -~ transplacental passage of maternal erythrocytes into the fetal circulation can be estimated by using the sickling phenomenon as a tracer. The maternal-fetal leakage of blood may exert a significant influence on the immunologic reactivity .of the infant. This is particularly important in the case of intrauterine exposure of the female Rh-negative fetus to Rh antigens from an Rh-positive mother. The purpose of this study is to demonstrate quantitatively transplacental passage of sickled maternal erythrocytes into the fetal circulation and to indicate that the maternofetal passage is as common as the fetomaternal passage of blood.
MATERIAL AND METHODS The placentas of patients known to have sicklemia were selected from Collaborative Study materials obtained from the University of Tennessee, Medical School Hospital, Memphis, Tennessee. The majority of the patients had sickle cell trait but hemoglobin electrophoreses were not routinely done. Only single live births, delivered spontaneously beyond 37 weeks of gestation and without maternal complications, were included in the study. For the proper calculation of sickling, only those showing a sickling rate of more than 90% in the intervillous space of the cotyledonary lobule were included.
Vol. 87, No. 5, pp. 781,783
782
Fujikura and Klionsky
The Journal of Pediatrics November 1975
4.1%. Small numbers of sickled erythrocytes were found in the fetal circulation of the placenta, especially in the umbilical veins. The incidence of concurrent sickling in maternal a n d fetal blood was 100% in the 44 cases selected. The mean sickling rate in umbilical or chorionic veins (8.4 _+ 5.1%) of 27 cases was higher than in those arteries (1.9 _+ 1.1%) of 25 cases. Furthermore, three out of 44 cases showed deported chorionic villi in umbilical or chorionic veins but not in those arteries. COMMENT
Fig. 1. Sickled erythrocytes and deported villi inlarge chorionic vein (x 225). This finding indicates fresh and mass contamination of maternal blood in the fetal circulation.
Table 1. Sickling rate of erythrocytes in the fetal and maternal circulation In tervillous space cotyledonary lobule
No.-of Cases examined Mean rate (%) SD
44 95.2 4.1
Umbilical or chorionic Vein
27 8.4 5.1
Arterv
25 1.9 1.1
Ten percent formalin was the best fixative for this purpose and the usual histopathologic methods of tissue processing preserved the sickled state of maternal erythrocytes. ~ The hematoxylin-eosin sections examined were from 3 cm wide blocks spanning the entire thickness of the placenta and from a cross section of the umbilical cord. If fetal erythrocytes were intact without artefacts, erythrocytes of umbilical or chorionic veins and arteries were evaluated for sickling counts. The m e a n sickling rate was calculated by counting the number of sickled erythrocytes out of a total of 500 erythrocytes in each area. In the 44 placentas the number of cases examined for sickling counts was limited to 25 of the artery and 27 of the vein due to various artefacts. RESULTS Table I shows the mean percentage a n d standard deviation of the sickling rate in three different areas: intervillous space, umbilical vein, and arteries. Since only cases showing a sickling rate of more than 90% were included in this study, the mean sickling rate in the intervillous space of the cotyledonary lobule was 95.2% _+
Third stage leakage. Maternofetal passage of the erythrocytes was found four times more frequently in umbilical or chorionic veins than in arteries. The higher percentage of sickled red blood cells in the veins emphasizes the acute nature of the maternofetal transfusion during the third stage of labor. Umbilical veins contain oxygenated blood (mean 31 mm Hg) returning from the placenta, whereas deoxygenated blood (mean 18 mm Hg) flows from fetus in the arteriesY In vitro analysis an oxygen tension of 45 mm Hg is taken to be the threshold for sickling of erythrocytes in patients with sickle cell anemia; the threshold for sickling in those with sickle cell trait is estimated to be 18 mm Hg. TM 11 If maternofetal passage of theerythrocytes occurs before the third stage of labor, t h e h i g h e r percentage of sickled cells could be found in umbilical or chorionic arteries where oxygen tensions are lower. The presence of deported chorionic villi in umbilical or chorionic veins indicates the possibility of a large shunt between the two circulations. When the placenta has separated, the common practice of placental expression may traumatize delicate chorionic villi anchoring the decidua. If the uterus is vigorously massaged or squeezed, such an attempt may increase the chance of villous damage. Sickling phenomenon. The mean sickling rate was 95.2% in the maternal circulation and 8.4% in umbilical or chorionic veinsl However, such a high sickling rate in vitro may not accurately reflect that of the in vivo phenomenon. It is unlikely that the high sickling rate actually occurs in vivo and may interfere with the placental circulation. If the sluggish flow of maternal erythrocytes does occur through the placenta, villous thrombosis and infarction would be increased, but these problems were not commonly found in the present study. Th e placental findings with sicklemia were not significantly different from those without sicklemia. The acidity of formalin fixative produces a right shift in the oxygen dissociation curve which exerts a favorable influence on the sickling phenomenon. 12 When the placenta separates from the uterine wall, the resulting
Volume 87 Number 5 hypoxia may create a natural sickling preparation of the trapped erythrocytes in the placenta. However, oxygenated erythrocytes in the decidual arteries of these 44 placentas were free from sickling, and deoxygenated erythrocytes only showed sickling in the maternal circulation. This indicates that the fixation and time intervals after delivery are not a strong influencing factor on the sickling p h e n o m e n o n of oxygenated erythrocytes in the n a t u r a l sickling preparation of the placenta. The presence of sickling based on sickling preparations has been reported in n e w b o r n infants. 6-8 However, these reports did not consider the possibility of the maternofetal passage and no appreciable quantity of S hemoglobin was detected by paper electrophoresis. The mechanism of sickling is obscure in newborn infants, since n e w b o r n blood contains about 70-80% of the fetal hemoglobin which is unable to produce sickling. The concentration of fetal hemoglobin in patients with sickle cell a n e m i a and sickle cell trait is known to be slightly high or in the normal range. 13 Placental transfusion. The blood volume of n e w b o r n infants varies over a wide range depending on the a m o u n t of placental transfusion at birth. Placental transfusion has been shown by an increase in weight of the infant of 55 to 180 gm during the first minutes of life when the umbilical cord is left unclamped. TM The volume of the placental transfusion has been estimated to be more than 40% above the initial infant blood 7olume when the umbilical cord is clamped immediately. TM Considering the large amount of placental transfusion during the third stage of labor, delayed ligation of the cord may increase the transplacental passage of maternal antigens into the fetal circulation and predispose the fetus to the development of immunologically mediated disease upon s u b s e q u e n t exposure to the antigen. 1~ D u n n and associates TM reported that the frequency of microtransfusions from fetus to mother occurring during the third stage of labor is not influenced by the timing of cord ligation. However, similar information on maternofetal transfusion is less well known in relation to the proper timing of cord ligation. This is particularly important in premature infants for providing an adequate blood volume without maternal blood contamination. Rh isoimmunization. Rh-positive mothers may induce immunologic tolerance or primary sensitization in their Rh-negative daughters as the result of transplacental passage of the maternal erythrocytes to their daughters in utero. However, this hypothesis of immunologic tolerance has not been supported b y factual evidence2 . . . . The effect of the grandmother's blood types on the incidence of erythroblastosis among their grandchildren was studied
Transplacental passage of maternal erythrocytes
7 83
by Taylor. 18 Of the children who had Rh-positive grandmothers, 62% were affected, whereas 46% of those with Rh-negative grandmothers were affected, an increase of 22%. 11 Thus there was a highly' statistically significant relation between the grandmother's Rh type and an increased incidence of erythroblastosis in the third generation. It is interesting to investigate whether Rh-negative female infants born of Rh-positive mothers should routinely be given prophylactic anti-D i m m u n e globulin at birth.~7 REFERENCES
1. Mengert WF, Rights CS, Bates CR, Reid AF, Wolf GR, and Nabors GC: Placental transmission of erythrocytes, Am J Obstet Gynecol, 69:678, 1955. 2. Macris NT, Hellman LM, and Watson RJ: The transmission of transfused sickle-trait cells from mother to fetus, Am J Obstet Gynecol 76:1214, 1958. 3. Hedenstedt S, and Naeslund J: Investigations of the permeability of the placenta with help of elliptocytes, Acta Med Scand [Suppl] 170:126, 1946. 4. Cohen F, and Zuelzer WW: The transplacental passage of maternal erythrocytes into the fetus, Obstet Gyneco193:566, 1965. 5. Fujikura T, and Froehlich L: Diagnosis of sickling by placental examination, Am J Obstet Gynecol 100:1122, 1968. 6. Porter FS, and Thurman WF: Studies of sickle cell disease, diagnosis in infancy, Am J Dis Child 106:35, 1963. 7. Schneider RG, and Haggard MD: Sickling, a quantitatively delayed genetic character, Proc Soc Exp Biol Med 89:196, 1955. 8. Watson J: The significance of the paucity of sickle cell cells in newborn Negro infants, Am J Med Sci 215:419, 1948. 9. Marquis L, and Ackerman BD: Placental respiration in the immediate neonatal period, Am J Obstet Gynecol 117:358, 1973. 10. Allison AC: Observations on the sickling phenomenon and on the distrbution of different hemoglobin types in erythrocyte populations, Clin Sci 15:497, 1950. 11. Sherman IJ: The sickling phenomenon with special reference to the differentiation of sickle celt anemia from the sickle cell trait, Bull Johns Hopkins Hosp 67:309, 1940. 12. Beutler E: Hypothesis: Changes in the oxygen dissociation curve and sickling: A general formulation and therapeutic strategy, Blood 43:297, 1974. 13. Ross Conference on Pediatric Research: Fetal hemoglobin, Columbus, Ohio, 1964, Ross Laboratories, pp 19 and 57. 14. Yao A, and Lind J: Placental transfusion, Am J Dis Child 127:128, 1974. 15. Scott JS: Immunological diseases and pregnancy, Br Med J 1;1559, 1966. 16. Dunn PM, Frazer ID, and Raper A: Influence of early cord ligation on the traneplacental passage of feotal cells, J Obstet Gynecol Br Commonw 73:757, 1966. 17. Scott. JR: Immunological factors in first pregnancy Rh isoimmunization, Lancet 1:717, 1973. 18. Taylor JF: Sensitization of Rh-negative daughters by their Rh-positive mothers, N Engl J Med 276:547, 1967.