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Transplacental passage of maternal erythrocytes
Transplacental Studies in early
JOHN
C.
CURTIS Rochester,
passage of maternal erythrocytes
pregnancy
DONOVAN, J.
New
LUND,
M.D. M.D.
York
TRANSPLACENTAL passage of maternal red cells is of fundamental importance to questions of immunologic competence and tolerance of the fetus. The literature to date records several studies limited to term pregnancies. Data presented in this paper are derived from the study of pregnancies between the twelfth and twenty-second weeks of amenorrhea.
Materials
and
weighed aliquot of incubated cells and weighing the amount injected. Details of laboratory procedure, isotope counting and calculations are presented in a previous paper.l However, it should be noted that the amount of sodium radiochromate used to incubate erythrocytes is about five times the amount used to incubate cells for a routine blood volume study and that the resultant count in the patient’s blood is also much higher. This was done to enhance the accuracy of the isotope technique as a device to detect placental transmission. The intervals between infusion of labeled cells and hysterotomy varied between 16 and 24 hours. All patients were ambulatory following infusion. A sample of circulating maternal blood was secured at the time of abortion for subsequent determination of its radioactivity. The products of conception were carefully handled so as to prevent or recognize possible contamination with maternal blood. This part of the technique is presented in detail because of its importance. The uterus was incised so as to expose but not rupture the gestational sac which was then dissected free and removed intact with the fetus contained therein. Each specimen was placed in a clean sink, maternal surface of the placenta downward, and gently rinsed under tap water for 2 or 3 minutes. Gloves were changed and the specimen was placed on a clean towel with the maternal surface of the placenta again downward. Most of the amniotic fluid was aspirated in a syringe by needle puncture of the membranes. Using
methods
The study group consists of 8 patients who were to have therapeutic abortions. Each subject had a normal pregnancy by all the usual criteria and the abortion was performed by abdominal hysterotomy. Vaginal abortions were not included because of the inevitable contamination with maternal blood. The series is not otherwise selected and represents all cases of abdominal hysterotomy performed in this department between January, 1962, and June? 1965. Maternal blood was incubated with 250 to 350 microcuries of sodium radiochromate and reinjected in each subject. Total counts injected were calculated by counting a
From the Department of Obstetrics and Gynecology, The University of Rochester School of Medicine and Dentistry. This inuestiqation tua~ supported Public Health Service Research HD-00274-3 from the National of Child Health and Human Development.
by Grant Institute
Presented at the Third Rochester Trophoblast Conference, Rochester, New York, Nov. I, 1965.
834
Transplacental
Table -__
passage
C:ase
Week
NO.
gestation 15 12 14 12 22 18 18 13
*Known
erythrocytes
835
I Maternal
: 3 4 5 6 7 8
of maternal
of
Total counts iniected
4,757,270 5.289,420 3.102,500 6.034,730 3.716,300 2,993.630 7.444,580 3,032,766
Amniotic
circulation
Fetus
(counts/min./
(counts/ min.)
ml.)
3652 5718 2554 4489 2580 2743 3574 2628
0 0 0 0 0 0 0 o/o
fluid (counts/
min.) 0 0 0 0
0 0 0
Cord blood fcounts/min.)
Cord (counts/ min.)
0 -0 0
----
6” 0 0 Cord and blood counted in toto-8”
0 0 10* Liver 0
0
contamination.
clean instruments, the partly collapsed membranes were opened between Allis clamps and the incised edges were held upward and slightly lateral. Gloves were changed, a clamp was inserted through the membrane incision, and the cord was divided after it had been clamped about 1 cm. from its placental insertion. The fetus and attached cord were placed in a clean basin after they had been lifted from the gestational sac without contact with maternal surfaces of either the placenta or membranes. Gloves were changed and the cord was unclamped and milked of all gross blood which was collected in a sterile tube containing an anticoagulant. The placental end of the cord was reclamped and the feta1 end was divided between clamps. The amputated cord, presumably with some trapped cells, was placed in a steri!e tube. Each fetal specimen was then wrapped in a sterile towel. Contamination was noted in 2 instances. This was recorded prospectively before the specimens were counted. Contamination of a third was recognized retrospectively. Collection of specimens after evacuation of the uterus required an estimated 4 to 6 minutes. There was no reason to suspect that any fetus had died prior to the final amputation of the cord. To check on this three of the fetal specimens were studied with EEG and ECG tracings after cord amputation. In each specimen so studied, electrical evidence of myocardial and central nervous system activity was present and seemed nor-
Table II. Dilution tagged with CP
to extinction
of red cells
1
I : 90,000 1 : 95.000 1: 100,000
4 0 0
0.0002 0.00015 0.0001
2
1: 90.000 1 : 95.000 1: 100.000
12 0 0
0.0002 0.00015 0.000 1
3
1: 90,000 1: 95,000 1: 100,000
23 16 0
0.0002 0.00015 0.0001
4
1 : 90,000 1 : 95.000 1:100000
13 8 0
0.0002 0.00015 0.000 1
ma1 although, admittedly, there are no adequate base lines as to what is normal at this stage of life. Specimens of maternal blood, amniotic fluid, cord blood, and cord were counted in a well-type scintillator with appropriate contro1s.l Total body counts were obtained for each fetal specimen. Counts expressed are those above background. The data are presented in Table I. To appraise the accuracy of the method, a sample of blood from each of four different patients was incubated with 250 to 350 microcuries of sodium radiochromate. A 1 ml. aliquot of packed cells from each sample was diluted serially. Each dilution was studied in the scintillator. Results are presented in Table II, together with the calculated
836
Donovan
and
Lund
equivalent in milliliters each dilution.
of tagged
cell> f’or
Observations
Each of the three positive counts is thought to result from contamination with maternal blood. Reasons derive from the examination of individual protocols. Contamination with maternal blood was noted in two specimens and recorded prior to counting. The cord blood (Case No. 5) and the cord (Case No. 7) are the two specimens. In the first instance, the placental end of the cord was clamped with an instrument contaminated with maternal blood. This was not recognized until after the cord blood had been milked past the contaminated end of the cord with gloves that had also handled the contaminated clamp. With the break in technique recognized, the cord was reclamped with clean instruments, the contaminated end removed, and the cord was rinsed between clamps for 20 minutes. When subsequently counted, the rinsed cord was negative as were the fetus and amniotic fluid in this case. In the second instance (cord, Case No. 7)) the surface of the cord was brushed accidentally across the maternal surface of the placenta after the cord had been milked of blood. The cord was deliberately not rinsed. A positive count was encountered although the cord blood in this case exhibited no count above background. The fetal specimen and amniotic fluid were likewise negative. The third instance of a positive count is Case No. 8. By error, the cord was not milked of blood and was sent to another laboratory together with the placental specimen. It is quite certain that the cord was handled with instruments used to handle the placenta. This was not appreciated until after the cord and clotted blood had been counted and discarded. As a further check in this case. the fetal liver was removed, emulsified, and studied in the scintillator counter. No evidence of radioactivity was detected. Comment
Mengert and associates’ have reported 4 cases of therapeutic abortion studied with
thy* preahortion infusioti nil l;E “-t:irg~d culls into the, ljaticnt. His rt~sults arr dificult to ei,aluatr sincca 3 of 4 co1 d blood spec iIllt,nh were negative while minimal radiation was detected in li of the 4 fetal specimens. using a method that was admittedlv unsatisfactor\ and vulnerable to error. The authors kno’w of no other data on maternal-fetal transmission of erythrocytes during early or rnidpregnancy. By contrast, the transfusion of term patients with isotope tagged or congenitally abnormal erythrocytes is a technique that has been employed by several investigators to study the phenomena of maternal-fetal transfusion. In most recorded studies the search for infused cells is limited to specimens of cord blood secured after vaginal delivery or during cesarcan section. Our experience indicates that the problem of surgical contamination with rnaternal blood has received inadequate attention in published results. Zarou, Lichtman, and Hellman” do not even mention this possible artifact. Macris, Hellman, and Watson4 state only that careful precautions were taken. Srnith and associates” state that precautions included copious rinsing of the cord but do not otherwise elaborate. In their studies, Naeslund and Nylin” indicate that the cut end of thcx cord was wiped beforc cord blood was collected. In our experience, this would be inadequate: the isotope method is so sensitive as to detect as little as 0.0002 ml. of tagged red ct:lls. Even minute contamination will ,give detectable counts and, if unrecognized. lead to crroneous conclusions. It is unfortunate that most studies have not included a sprcimcn of peripheral blood secured from the infant a few hours after delivery. Infant blood secured by heel puncture was studied by Hedenstedt and -Naeslund7 who transfused 2 term patients with 200 to 300 c.c. of elliptocytic blood. One infant. born of a normal mother after a normal labor and delivery, showed no elliptocytes in the peripheral blood. The second infant was found to exhibit a 27 per cent elliptocyte count with a 36 per cent count in the mother. The child‘s count fell to normal in .5 days. ‘The
Transplacental
patient in this case was toxic and the placenta revealed numerous infarcts and recent hemorrhages. Mengert and associates,? later employed a similar technique in 2 term pregnancies. He seems to have excluded surgical contamination by appearing to demonstrate sickle cells in stained sections of the cord in one or two cases studied. He points out the difficulty of distinguishing between an occasional sickled erythrocyte and a norma red cell in a fixed section. After this paper had been prepared for presentation, the excellent study of Cohen and Zuelze? was published. Using an immunofluorescent technique and studying infant heel and cord blood in term cases, they conclude that the reported passage of maternal red cells into the fetal circulation reflects unavoidable contamination of cord blood. They believe that passage of maternal erythrocytes into the fetus is an exceptional occurrence.
2.
3. 4. 5.
Donovan, J. C., Lund, C. J., and Whalen, L.: Surg. Obst. & Gynec. 119: 1031, 1964. Mengert, W. F., Rights, C. S., Bates, C. R., Jr., Reid, A. F., Wolf, G. R., and Nabors, G. C.: AM. J. OBST. & GYNEC. 69: 678, 1955. Zarou, D. M., Lichtman, H. C., and Hellman, L. M.: AM. T. OBST. & GYNEC. 88: 565. 1964. Macris, N. ?., Hellman, L. M., and Gatson, R. J.: AM. J. OBST. & GYNEC. 76: 1214, 1958. Smith, K., Duhring, J. L., Greene, J. W.,
of maternal
erythrocytes
837
Summary Crsl-tagged erythrocytes were injected in 8 women with normal pregnancies who were scheduled for abdominal hysterotomy between the twelfth and twenty-second weeks of gestation. Easily followed but careful precautions against contamination with maternal blood are described. No evidence of radioactivity was found except in 3 isolated specimens in which known contamination had occurred due to human error. It is concluded that the study has failed to demonstrate maternal-fetal transfusion in 8 cases of early pregnancy. Dilution studies are cited to indicate the method described is sufficiently sensitive as to detect as little as 0.0002 ml. of tagged cells. Attention is called to the likelihood of contamination in term studies that are limited to the investigation of umbilical cord blood.
REFERENCES
1.
passage
6. 7. 8.
Rochlin, D. B., and Blakemore, W. S.: Obst. & Gynec. 18: 673, 1961. Naeslund, J., and Nylin, G.: Acta med. scandinav. (Suppl.) 170: 390, 1946. Hedenstedt, S., and Naeslund, J.: Acta med. scandinav. (Suppl.) 170: 126, 1946. Cohen, F., and Zuelzer, W. W.: AM. J. OBST. & GYNEC. 93: 566, 1965. 260 Crittenden Boulevard Rochester, New York 14620
JOHN
C.
CURTIS Rochester,
passage of maternal erythrocytes
pregnancy
DONOVAN, J.
New
LUND,
M.D. M.D.
York
TRANSPLACENTAL passage of maternal red cells is of fundamental importance to questions of immunologic competence and tolerance of the fetus. The literature to date records several studies limited to term pregnancies. Data presented in this paper are derived from the study of pregnancies between the twelfth and twenty-second weeks of amenorrhea.
Materials
and
weighed aliquot of incubated cells and weighing the amount injected. Details of laboratory procedure, isotope counting and calculations are presented in a previous paper.l However, it should be noted that the amount of sodium radiochromate used to incubate erythrocytes is about five times the amount used to incubate cells for a routine blood volume study and that the resultant count in the patient’s blood is also much higher. This was done to enhance the accuracy of the isotope technique as a device to detect placental transmission. The intervals between infusion of labeled cells and hysterotomy varied between 16 and 24 hours. All patients were ambulatory following infusion. A sample of circulating maternal blood was secured at the time of abortion for subsequent determination of its radioactivity. The products of conception were carefully handled so as to prevent or recognize possible contamination with maternal blood. This part of the technique is presented in detail because of its importance. The uterus was incised so as to expose but not rupture the gestational sac which was then dissected free and removed intact with the fetus contained therein. Each specimen was placed in a clean sink, maternal surface of the placenta downward, and gently rinsed under tap water for 2 or 3 minutes. Gloves were changed and the specimen was placed on a clean towel with the maternal surface of the placenta again downward. Most of the amniotic fluid was aspirated in a syringe by needle puncture of the membranes. Using
methods
The study group consists of 8 patients who were to have therapeutic abortions. Each subject had a normal pregnancy by all the usual criteria and the abortion was performed by abdominal hysterotomy. Vaginal abortions were not included because of the inevitable contamination with maternal blood. The series is not otherwise selected and represents all cases of abdominal hysterotomy performed in this department between January, 1962, and June? 1965. Maternal blood was incubated with 250 to 350 microcuries of sodium radiochromate and reinjected in each subject. Total counts injected were calculated by counting a
From the Department of Obstetrics and Gynecology, The University of Rochester School of Medicine and Dentistry. This inuestiqation tua~ supported Public Health Service Research HD-00274-3 from the National of Child Health and Human Development.
by Grant Institute
Presented at the Third Rochester Trophoblast Conference, Rochester, New York, Nov. I, 1965.
834
Transplacental
Table -__
passage
C:ase
Week
NO.
gestation 15 12 14 12 22 18 18 13
*Known
erythrocytes
835
I Maternal
: 3 4 5 6 7 8
of maternal
of
Total counts iniected
4,757,270 5.289,420 3.102,500 6.034,730 3.716,300 2,993.630 7.444,580 3,032,766
Amniotic
circulation
Fetus
(counts/min./
(counts/ min.)
ml.)
3652 5718 2554 4489 2580 2743 3574 2628
0 0 0 0 0 0 0 o/o
fluid (counts/
min.) 0 0 0 0
0 0 0
Cord blood fcounts/min.)
Cord (counts/ min.)
0 -0 0
----
6” 0 0 Cord and blood counted in toto-8”
0 0 10* Liver 0
0
contamination.
clean instruments, the partly collapsed membranes were opened between Allis clamps and the incised edges were held upward and slightly lateral. Gloves were changed, a clamp was inserted through the membrane incision, and the cord was divided after it had been clamped about 1 cm. from its placental insertion. The fetus and attached cord were placed in a clean basin after they had been lifted from the gestational sac without contact with maternal surfaces of either the placenta or membranes. Gloves were changed and the cord was unclamped and milked of all gross blood which was collected in a sterile tube containing an anticoagulant. The placental end of the cord was reclamped and the feta1 end was divided between clamps. The amputated cord, presumably with some trapped cells, was placed in a steri!e tube. Each fetal specimen was then wrapped in a sterile towel. Contamination was noted in 2 instances. This was recorded prospectively before the specimens were counted. Contamination of a third was recognized retrospectively. Collection of specimens after evacuation of the uterus required an estimated 4 to 6 minutes. There was no reason to suspect that any fetus had died prior to the final amputation of the cord. To check on this three of the fetal specimens were studied with EEG and ECG tracings after cord amputation. In each specimen so studied, electrical evidence of myocardial and central nervous system activity was present and seemed nor-
Table II. Dilution tagged with CP
to extinction
of red cells
1
I : 90,000 1 : 95.000 1: 100,000
4 0 0
0.0002 0.00015 0.0001
2
1: 90.000 1 : 95.000 1: 100.000
12 0 0
0.0002 0.00015 0.000 1
3
1: 90,000 1: 95,000 1: 100,000
23 16 0
0.0002 0.00015 0.0001
4
1 : 90,000 1 : 95.000 1:100000
13 8 0
0.0002 0.00015 0.000 1
ma1 although, admittedly, there are no adequate base lines as to what is normal at this stage of life. Specimens of maternal blood, amniotic fluid, cord blood, and cord were counted in a well-type scintillator with appropriate contro1s.l Total body counts were obtained for each fetal specimen. Counts expressed are those above background. The data are presented in Table I. To appraise the accuracy of the method, a sample of blood from each of four different patients was incubated with 250 to 350 microcuries of sodium radiochromate. A 1 ml. aliquot of packed cells from each sample was diluted serially. Each dilution was studied in the scintillator. Results are presented in Table II, together with the calculated
836
Donovan
and
Lund
equivalent in milliliters each dilution.
of tagged
cell> f’or
Observations
Each of the three positive counts is thought to result from contamination with maternal blood. Reasons derive from the examination of individual protocols. Contamination with maternal blood was noted in two specimens and recorded prior to counting. The cord blood (Case No. 5) and the cord (Case No. 7) are the two specimens. In the first instance, the placental end of the cord was clamped with an instrument contaminated with maternal blood. This was not recognized until after the cord blood had been milked past the contaminated end of the cord with gloves that had also handled the contaminated clamp. With the break in technique recognized, the cord was reclamped with clean instruments, the contaminated end removed, and the cord was rinsed between clamps for 20 minutes. When subsequently counted, the rinsed cord was negative as were the fetus and amniotic fluid in this case. In the second instance (cord, Case No. 7)) the surface of the cord was brushed accidentally across the maternal surface of the placenta after the cord had been milked of blood. The cord was deliberately not rinsed. A positive count was encountered although the cord blood in this case exhibited no count above background. The fetal specimen and amniotic fluid were likewise negative. The third instance of a positive count is Case No. 8. By error, the cord was not milked of blood and was sent to another laboratory together with the placental specimen. It is quite certain that the cord was handled with instruments used to handle the placenta. This was not appreciated until after the cord and clotted blood had been counted and discarded. As a further check in this case. the fetal liver was removed, emulsified, and studied in the scintillator counter. No evidence of radioactivity was detected. Comment
Mengert and associates’ have reported 4 cases of therapeutic abortion studied with
thy* preahortion infusioti nil l;E “-t:irg~d culls into the, ljaticnt. His rt~sults arr dificult to ei,aluatr sincca 3 of 4 co1 d blood spec iIllt,nh were negative while minimal radiation was detected in li of the 4 fetal specimens. using a method that was admittedlv unsatisfactor\ and vulnerable to error. The authors kno’w of no other data on maternal-fetal transmission of erythrocytes during early or rnidpregnancy. By contrast, the transfusion of term patients with isotope tagged or congenitally abnormal erythrocytes is a technique that has been employed by several investigators to study the phenomena of maternal-fetal transfusion. In most recorded studies the search for infused cells is limited to specimens of cord blood secured after vaginal delivery or during cesarcan section. Our experience indicates that the problem of surgical contamination with rnaternal blood has received inadequate attention in published results. Zarou, Lichtman, and Hellman” do not even mention this possible artifact. Macris, Hellman, and Watson4 state only that careful precautions were taken. Srnith and associates” state that precautions included copious rinsing of the cord but do not otherwise elaborate. In their studies, Naeslund and Nylin” indicate that the cut end of thcx cord was wiped beforc cord blood was collected. In our experience, this would be inadequate: the isotope method is so sensitive as to detect as little as 0.0002 ml. of tagged red ct:lls. Even minute contamination will ,give detectable counts and, if unrecognized. lead to crroneous conclusions. It is unfortunate that most studies have not included a sprcimcn of peripheral blood secured from the infant a few hours after delivery. Infant blood secured by heel puncture was studied by Hedenstedt and -Naeslund7 who transfused 2 term patients with 200 to 300 c.c. of elliptocytic blood. One infant. born of a normal mother after a normal labor and delivery, showed no elliptocytes in the peripheral blood. The second infant was found to exhibit a 27 per cent elliptocyte count with a 36 per cent count in the mother. The child‘s count fell to normal in .5 days. ‘The
Transplacental
patient in this case was toxic and the placenta revealed numerous infarcts and recent hemorrhages. Mengert and associates,? later employed a similar technique in 2 term pregnancies. He seems to have excluded surgical contamination by appearing to demonstrate sickle cells in stained sections of the cord in one or two cases studied. He points out the difficulty of distinguishing between an occasional sickled erythrocyte and a norma red cell in a fixed section. After this paper had been prepared for presentation, the excellent study of Cohen and Zuelze? was published. Using an immunofluorescent technique and studying infant heel and cord blood in term cases, they conclude that the reported passage of maternal red cells into the fetal circulation reflects unavoidable contamination of cord blood. They believe that passage of maternal erythrocytes into the fetus is an exceptional occurrence.
2.
3. 4. 5.
Donovan, J. C., Lund, C. J., and Whalen, L.: Surg. Obst. & Gynec. 119: 1031, 1964. Mengert, W. F., Rights, C. S., Bates, C. R., Jr., Reid, A. F., Wolf, G. R., and Nabors, G. C.: AM. J. OBST. & GYNEC. 69: 678, 1955. Zarou, D. M., Lichtman, H. C., and Hellman, L. M.: AM. T. OBST. & GYNEC. 88: 565. 1964. Macris, N. ?., Hellman, L. M., and Gatson, R. J.: AM. J. OBST. & GYNEC. 76: 1214, 1958. Smith, K., Duhring, J. L., Greene, J. W.,
of maternal
erythrocytes
837
Summary Crsl-tagged erythrocytes were injected in 8 women with normal pregnancies who were scheduled for abdominal hysterotomy between the twelfth and twenty-second weeks of gestation. Easily followed but careful precautions against contamination with maternal blood are described. No evidence of radioactivity was found except in 3 isolated specimens in which known contamination had occurred due to human error. It is concluded that the study has failed to demonstrate maternal-fetal transfusion in 8 cases of early pregnancy. Dilution studies are cited to indicate the method described is sufficiently sensitive as to detect as little as 0.0002 ml. of tagged cells. Attention is called to the likelihood of contamination in term studies that are limited to the investigation of umbilical cord blood.
REFERENCES
1.
passage
6. 7. 8.
Rochlin, D. B., and Blakemore, W. S.: Obst. & Gynec. 18: 673, 1961. Naeslund, J., and Nylin, G.: Acta med. scandinav. (Suppl.) 170: 390, 1946. Hedenstedt, S., and Naeslund, J.: Acta med. scandinav. (Suppl.) 170: 126, 1946. Cohen, F., and Zuelzer, W. W.: AM. J. OBST. & GYNEC. 93: 566, 1965. 260 Crittenden Boulevard Rochester, New York 14620