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Transplacental hemorrhage in cesarean section
Transplacental hemorrhage in cesarean section ANDREW C. W. MONTAGUE, M . D.* JULIUS R. KREVANS, M.D . Baltimore , Maryland
l' H E P 0 S SIB L E role of transplacental hemorrhage (TPH) in Rh isoimmunization was suspected by Levine Katz, and Burnham! in 1941. In 1948, Wiener2 described a case of anemia in a fetus whose Rh-negative mother had developed antibodies in the third trimester of pregnancy. Wiener postulated that immunization was secondary to a transplacental hemorrhage. Borst-Eilers3 demonstrated fetal cells in maternal circulation by the differential agglutination method in 1961. Since then other methods have been devised for detection of fetal cells in the maternal circulation and the role of transplacental hemorrhage as a cause of Rh isoimmunization has been firmly established. In 1963, Finn, Harper, and Stallings4 described their experiments with the Kleihauer elution technique 5 and proved its sensitivity to detect fetal cells in the dilution in the maternal blood up to 1 to 5,000. Their results were not entirely quantitative. Zipurski and associates,6, 7. 8 using a similar method, were able to detect transplacental hemorrhage of the order of 0.1 c.c. They have studied 204 patients with complicated pregnancies and report that at cesarean section 15 per cent of the patients showed evidence of transplacental hemorrhage. To contribute some direct measurements
of the amount of transplacental hemorrhage, we have studied a group of patients who underwent cesarean section on the obstetrical services of The Johns Hopkins Hospital and Baltimore City Hospitals. Material and methods
A modification of the Kleihauer acid elution technique 9 for detection of fetal cells in maternal circulation was used. Thin, undiluted smears were made using heparinanticoagulated blood. Smears are allowed to dry and are fixed for 5 minutes in 80 per cent ethanol. Then slides are washed with tap water and air dried. A citric acid phosphate buffer (to 53 c.c. of 0.1M citric acid solution, add 147 C.c. of 0.2M Na 2HP0 4 ) was freshly prepared. Proper pH of this buffer is essential and should be at 3.3. Before use, the solution was warmed for one hour to the temperature of 37° C. and all the possible bubbles were eliminated. The previously fixed blood smears were immersed in the buffer for 5 minutes, then washed with tap water and air dried. Slides were then stained for 4 minutes with Meyer's hematoxylin and, following another wash with distilled water and air drying, were counterstained for 4 minutes with 0.1 per cent aqueous solution of erythrosin. During the above-described procedure, adult hemoglobin is eluted from maternal erythrocytes leaving fetal cells stained strongly pink and easily distinguishable from pale "ghost" maternal cells. Quantification of the m ethod. The number of fetal red blood cells per high-power field varies with many factors such as thick-
From The Department of Gynecology and Obstetrics, The Johns Hopkins University School of Medicine, and The Department of Medicine, Baltimore City Hospitals. Supported in part by United States Public Health Service Grant 2TI-HD23-04. * Josiah Macy, Jr . Foundation Fellow.
1115
1116
August 15, 1966
Montague and Krevans
Am .
ness of smear, maternal hematocrit, etc., and it is technically difficult to count the percentage of fetal red blood cells to maternal red blood cells. We have elected to count fetal red blood cells as the ratio of white blood cells on the smear. (The white blood count of maternal sample is determined simultaneously, and our ratio is corrected to a hypothetical white blood count of 10,000 per cubic millimeter ) . To determine the fctal red blood cell/ white blood ceIl ratio, we simply count 1,000 white blood cells and include all the fetal red blood cells encountered in the above count. A skilled technician can take less than 10 minutes to count the entire slide. Calibration of the method. To test the accuracy and precision of our method we have injected, in a manner similar to that of Finn and associates,lO, 11 known amounts of fetal red blood cells into Rh and ABO compatible volunteers. A preinjection blood sample was taken from each subject, then the fetal blood was injected intravenously and, half an hour later, venous samples were drawn from the opposite arm.
Table I. Correlation between amounts of blood injected and corrected fetal red blood cell-maternal white blood cell ratio - - - -. _-._ ...
Amount in iected (c.c) 0.25 0.25 0.25 0.25 0.25 0.5 D.5 0.5 0.5 1.0 1.0
1.0 1.0 1.0 5.0 5.0 5.0 10.0 10.0 10.0 10.0
. Corrected (FRBC 11,000 WBC) 25) 251 25r 2+1 25J 621 5:1l 57r 60J 116) 1331 102r 1261 137 J 653)
547r
400J 1,02 31 851l 959r 700J
I
Preinjection (FRBCII,OOO WBC) 9 10 4 6 6
J. Obs!. & Gynec.
The placental blood was anticoagulated with ACD and used within 2 hours from the end of the third stage of labor. The results of these injections are given in Table 1. Kearly all the male volunteers have shown corrected counts of 5 to 19 deeply stained cells per 1,000 white blood cells prior to injection of placental blood. We are faced with either false positives, red-stained impurities, or with naturally occurring fetal hemoglobin erythrocytes among normal males without evidence of hemoglobinopathies (Fig. 1). ~o false negatives were seen in our series of 21 male subjects. Table I shows that our method is most accurate in the injection range of 0.25 C.c. to 1 C.c. of placental blood. That is why we have selected it to study the amount of transplacental hemorrhage in cesarean sections which was previously described by Zipurski to be in the same range (Table II \ . Experimental results
For our study we used 30 patients who were undergoing cesarean section or cesarean section hysterectomy in The Johns Hopkins Hospital and in the Baltimore City Hospitals. Patients were not selected and were taken in consecutive order. The clinical features of each case were noted, but the laboratory was unaware of either the clinical history or the white blood count correction factor at the time when feta l red blood cell per 1,000 white blood cell counts were made. The amount of transplacental hemorrhage was estimated by using a curve derived from Fig. 1. Preoperative and half-hour postoperative samples of maternal blood were taken and
Table II. Recovery of injected fetal cells 19 14 16 5 9 5
Volume of fetal blood injected (c. c.) 10 5 1 0.5 0.25
No. of subjects 4 3
5
4 5
Fetal RBC demonstrated after injection -I-
3 5
4 .'i
. ..
_--
Volume 95 Number 8
Transplacental hemorrhage
140
130 120
-m 110 ~ 100 0 0
0
2 90 ..... 0 m 80
II::
70
'0
60
. c:; .......
IL
0
0
50 40 30 20
.....
10 0.25
0.5
1.0
Vol. of Fetol Blood Injecte d in mi.
Fig. 1.
two slides were made of each blood sample. White blood cells were determined by using a Coulter counter. The amount of transplacental hemorrhage caused by cesarean section was taken to be the difference between the amount of fetal red blood cells before and after the section. That method allowed us to evaluate the influence of other possible factors, such as presence of labor, etc., which might have influenced the amount of transplacental hemorrhage. It will be noted that 21 out of 30 patients showed evidence of transplacental hemorrhage smaller or equal to 0.25 C.c. which agrees with the findings of Zipurski. The problem whether our estimations of the amount of transplacental hemorrhage apply also in ABO incompatible pregnancies worried us and, in an effort to analyze our Table III. Correlation between amount of transplacental hemorrhage and ABO compatibility ABO compatible pregnancies ABO incompatible ABO compatibility unknown
<0.1
10.1>0.2491
0.25+
9
6 1
4
2
2
2 3
1117
data, we went back and obtained the fetal blood group in 22 out of 30 patients. Table III shows the amount of transplacental hemorrhage in compatible, incompatible, and unknown pregnancies. We were unable to determine whether the amount of transplacental hemorrhage obtained by our method in ABO incompatible pregnancies is reliable, but errors should only be in the direction of underestimating the amount of transplacental hemorrhage. It seems clear from Table IV that the presence or absence of labor influences the transplacental hemorrhage associated with cesarean section. Furthermore, all but one of the 8 cases of cesarean section complicated by other obstetrical factors had transplacental hemorrhage larger than 0.25 c.c. Our figures support the hypothesis that the amount of transplacental hemorrhage is determined less by a given obstetrical procedure than by the condition which was the indication for that procedure (Table V). It is interesting to notice that the largest transplacental hemorrhage in our series occurred in a patient who had abruptio placentae and that the subsequent cesarean section did not materially alter the amount of the transplacental hemorrhage. The data presented above do not support the idea that cesarean section should be Table IV. Effect of labor on amount of transplacental hemorrhage Size
Labor
No labor
0.1 0.1 to 0.25 0.25+
1 3
12 5 2
7
Table V. Effect of obstetrical complications on amount of transplacental hemorrhage Type of complication Breech Twins Transverse lie Adherent placenta Placenta previa Abruptio placentae
Total No. 3 1 1 2 1 1
Under lover 0.25 C.c. 0.20 c.c. 2 1 1 2 1 1
1118
Montague and Krevans
avoided in unsensitized Rh-negative patients, but if a cesarean section is to be performed, it would be preferable to avoid labor as a superimposing factor in causing transplacental hemorrhage. Summary
A modification of the Finn's method of quantitatively estimating small transplacental hemorrhage IS presented and discussed.
August 15, 1966 Am, J, Obl;t. & Gynec.
Amount of transplacental hemorrhage caused by cesarean section and cesarean section hysterectomy is estimated in 30 cases, showing that 21 out of 30 in our series have a transplacental hemorrhage equal to or less than 0.25 c.c. The clinical history of the patients and their surgical procedures are correlated with the amount of transplacental hemorrhage.
REFERENCES
1. Levine, P., Katz, N. E. M., and Burnham, L.: l A. M. A. 116: 825, 1941. 2. Wiener, A. S.: AM. l OBST. & GYXEC. 56: 717, 1948. 3. Borst-Eilers, E.: Vox Sang. 6: 451, 1961. 4. Finn, R., Harper, D. T., and Stallings, S. A.: Transfusion 3: 114, 1963. 5. Kleihauer, E., Braun, H., and Betke, K.: Klin. Wchnschr. 35: 637, 1957. 6. Zipurski, A., Hull, A., and White, F. D.: Lancet 1: 451, 1959. 7. Zipurski, A., Pollack, l, Neelands, P., Chown,
B., and Israels, L. G.: Lancet 2: 489, 1963. 8. Zipurski, A., Pollack, J., Chown, B., and Israels, L. G.: Lancet 2: 493, 1963. 9. Kleihauer, E., and Betke, K.: Internist 1: 292, 1960. 10. Finn, R., Clarke, C. A., Donohoe, W. T. A., McConnell, R. B., Sheppard, P. M., Lehane, D., and Kulke, W.: Brit. M. l 1: 1486, 1961. 11. Clarke, C. A., Donnohoe, W. T. A., McConnell, R. B., Woodrow, l C., Finn, R., Krevans, l R., Kulke, W., Lehane, D., and Sheppard, P. M.: Brit. M. l 1: 979, 1963.
l' H E P 0 S SIB L E role of transplacental hemorrhage (TPH) in Rh isoimmunization was suspected by Levine Katz, and Burnham! in 1941. In 1948, Wiener2 described a case of anemia in a fetus whose Rh-negative mother had developed antibodies in the third trimester of pregnancy. Wiener postulated that immunization was secondary to a transplacental hemorrhage. Borst-Eilers3 demonstrated fetal cells in maternal circulation by the differential agglutination method in 1961. Since then other methods have been devised for detection of fetal cells in the maternal circulation and the role of transplacental hemorrhage as a cause of Rh isoimmunization has been firmly established. In 1963, Finn, Harper, and Stallings4 described their experiments with the Kleihauer elution technique 5 and proved its sensitivity to detect fetal cells in the dilution in the maternal blood up to 1 to 5,000. Their results were not entirely quantitative. Zipurski and associates,6, 7. 8 using a similar method, were able to detect transplacental hemorrhage of the order of 0.1 c.c. They have studied 204 patients with complicated pregnancies and report that at cesarean section 15 per cent of the patients showed evidence of transplacental hemorrhage. To contribute some direct measurements
of the amount of transplacental hemorrhage, we have studied a group of patients who underwent cesarean section on the obstetrical services of The Johns Hopkins Hospital and Baltimore City Hospitals. Material and methods
A modification of the Kleihauer acid elution technique 9 for detection of fetal cells in maternal circulation was used. Thin, undiluted smears were made using heparinanticoagulated blood. Smears are allowed to dry and are fixed for 5 minutes in 80 per cent ethanol. Then slides are washed with tap water and air dried. A citric acid phosphate buffer (to 53 c.c. of 0.1M citric acid solution, add 147 C.c. of 0.2M Na 2HP0 4 ) was freshly prepared. Proper pH of this buffer is essential and should be at 3.3. Before use, the solution was warmed for one hour to the temperature of 37° C. and all the possible bubbles were eliminated. The previously fixed blood smears were immersed in the buffer for 5 minutes, then washed with tap water and air dried. Slides were then stained for 4 minutes with Meyer's hematoxylin and, following another wash with distilled water and air drying, were counterstained for 4 minutes with 0.1 per cent aqueous solution of erythrosin. During the above-described procedure, adult hemoglobin is eluted from maternal erythrocytes leaving fetal cells stained strongly pink and easily distinguishable from pale "ghost" maternal cells. Quantification of the m ethod. The number of fetal red blood cells per high-power field varies with many factors such as thick-
From The Department of Gynecology and Obstetrics, The Johns Hopkins University School of Medicine, and The Department of Medicine, Baltimore City Hospitals. Supported in part by United States Public Health Service Grant 2TI-HD23-04. * Josiah Macy, Jr . Foundation Fellow.
1115
1116
August 15, 1966
Montague and Krevans
Am .
ness of smear, maternal hematocrit, etc., and it is technically difficult to count the percentage of fetal red blood cells to maternal red blood cells. We have elected to count fetal red blood cells as the ratio of white blood cells on the smear. (The white blood count of maternal sample is determined simultaneously, and our ratio is corrected to a hypothetical white blood count of 10,000 per cubic millimeter ) . To determine the fctal red blood cell/ white blood ceIl ratio, we simply count 1,000 white blood cells and include all the fetal red blood cells encountered in the above count. A skilled technician can take less than 10 minutes to count the entire slide. Calibration of the method. To test the accuracy and precision of our method we have injected, in a manner similar to that of Finn and associates,lO, 11 known amounts of fetal red blood cells into Rh and ABO compatible volunteers. A preinjection blood sample was taken from each subject, then the fetal blood was injected intravenously and, half an hour later, venous samples were drawn from the opposite arm.
Table I. Correlation between amounts of blood injected and corrected fetal red blood cell-maternal white blood cell ratio - - - -. _-._ ...
Amount in iected (c.c) 0.25 0.25 0.25 0.25 0.25 0.5 D.5 0.5 0.5 1.0 1.0
1.0 1.0 1.0 5.0 5.0 5.0 10.0 10.0 10.0 10.0
. Corrected (FRBC 11,000 WBC) 25) 251 25r 2+1 25J 621 5:1l 57r 60J 116) 1331 102r 1261 137 J 653)
547r
400J 1,02 31 851l 959r 700J
I
Preinjection (FRBCII,OOO WBC) 9 10 4 6 6
J. Obs!. & Gynec.
The placental blood was anticoagulated with ACD and used within 2 hours from the end of the third stage of labor. The results of these injections are given in Table 1. Kearly all the male volunteers have shown corrected counts of 5 to 19 deeply stained cells per 1,000 white blood cells prior to injection of placental blood. We are faced with either false positives, red-stained impurities, or with naturally occurring fetal hemoglobin erythrocytes among normal males without evidence of hemoglobinopathies (Fig. 1). ~o false negatives were seen in our series of 21 male subjects. Table I shows that our method is most accurate in the injection range of 0.25 C.c. to 1 C.c. of placental blood. That is why we have selected it to study the amount of transplacental hemorrhage in cesarean sections which was previously described by Zipurski to be in the same range (Table II \ . Experimental results
For our study we used 30 patients who were undergoing cesarean section or cesarean section hysterectomy in The Johns Hopkins Hospital and in the Baltimore City Hospitals. Patients were not selected and were taken in consecutive order. The clinical features of each case were noted, but the laboratory was unaware of either the clinical history or the white blood count correction factor at the time when feta l red blood cell per 1,000 white blood cell counts were made. The amount of transplacental hemorrhage was estimated by using a curve derived from Fig. 1. Preoperative and half-hour postoperative samples of maternal blood were taken and
Table II. Recovery of injected fetal cells 19 14 16 5 9 5
Volume of fetal blood injected (c. c.) 10 5 1 0.5 0.25
No. of subjects 4 3
5
4 5
Fetal RBC demonstrated after injection -I-
3 5
4 .'i
. ..
_--
Volume 95 Number 8
Transplacental hemorrhage
140
130 120
-m 110 ~ 100 0 0
0
2 90 ..... 0 m 80
II::
70
'0
60
. c:; .......
IL
0
0
50 40 30 20
.....
10 0.25
0.5
1.0
Vol. of Fetol Blood Injecte d in mi.
Fig. 1.
two slides were made of each blood sample. White blood cells were determined by using a Coulter counter. The amount of transplacental hemorrhage caused by cesarean section was taken to be the difference between the amount of fetal red blood cells before and after the section. That method allowed us to evaluate the influence of other possible factors, such as presence of labor, etc., which might have influenced the amount of transplacental hemorrhage. It will be noted that 21 out of 30 patients showed evidence of transplacental hemorrhage smaller or equal to 0.25 C.c. which agrees with the findings of Zipurski. The problem whether our estimations of the amount of transplacental hemorrhage apply also in ABO incompatible pregnancies worried us and, in an effort to analyze our Table III. Correlation between amount of transplacental hemorrhage and ABO compatibility ABO compatible pregnancies ABO incompatible ABO compatibility unknown
<0.1
10.1>0.2491
0.25+
9
6 1
4
2
2
2 3
1117
data, we went back and obtained the fetal blood group in 22 out of 30 patients. Table III shows the amount of transplacental hemorrhage in compatible, incompatible, and unknown pregnancies. We were unable to determine whether the amount of transplacental hemorrhage obtained by our method in ABO incompatible pregnancies is reliable, but errors should only be in the direction of underestimating the amount of transplacental hemorrhage. It seems clear from Table IV that the presence or absence of labor influences the transplacental hemorrhage associated with cesarean section. Furthermore, all but one of the 8 cases of cesarean section complicated by other obstetrical factors had transplacental hemorrhage larger than 0.25 c.c. Our figures support the hypothesis that the amount of transplacental hemorrhage is determined less by a given obstetrical procedure than by the condition which was the indication for that procedure (Table V). It is interesting to notice that the largest transplacental hemorrhage in our series occurred in a patient who had abruptio placentae and that the subsequent cesarean section did not materially alter the amount of the transplacental hemorrhage. The data presented above do not support the idea that cesarean section should be Table IV. Effect of labor on amount of transplacental hemorrhage Size
Labor
No labor
0.1 0.1 to 0.25 0.25+
1 3
12 5 2
7
Table V. Effect of obstetrical complications on amount of transplacental hemorrhage Type of complication Breech Twins Transverse lie Adherent placenta Placenta previa Abruptio placentae
Total No. 3 1 1 2 1 1
Under lover 0.25 C.c. 0.20 c.c. 2 1 1 2 1 1
1118
Montague and Krevans
avoided in unsensitized Rh-negative patients, but if a cesarean section is to be performed, it would be preferable to avoid labor as a superimposing factor in causing transplacental hemorrhage. Summary
A modification of the Finn's method of quantitatively estimating small transplacental hemorrhage IS presented and discussed.
August 15, 1966 Am, J, Obl;t. & Gynec.
Amount of transplacental hemorrhage caused by cesarean section and cesarean section hysterectomy is estimated in 30 cases, showing that 21 out of 30 in our series have a transplacental hemorrhage equal to or less than 0.25 c.c. The clinical history of the patients and their surgical procedures are correlated with the amount of transplacental hemorrhage.
REFERENCES
1. Levine, P., Katz, N. E. M., and Burnham, L.: l A. M. A. 116: 825, 1941. 2. Wiener, A. S.: AM. l OBST. & GYXEC. 56: 717, 1948. 3. Borst-Eilers, E.: Vox Sang. 6: 451, 1961. 4. Finn, R., Harper, D. T., and Stallings, S. A.: Transfusion 3: 114, 1963. 5. Kleihauer, E., Braun, H., and Betke, K.: Klin. Wchnschr. 35: 637, 1957. 6. Zipurski, A., Hull, A., and White, F. D.: Lancet 1: 451, 1959. 7. Zipurski, A., Pollack, l, Neelands, P., Chown,
B., and Israels, L. G.: Lancet 2: 489, 1963. 8. Zipurski, A., Pollack, J., Chown, B., and Israels, L. G.: Lancet 2: 493, 1963. 9. Kleihauer, E., and Betke, K.: Internist 1: 292, 1960. 10. Finn, R., Clarke, C. A., Donohoe, W. T. A., McConnell, R. B., Sheppard, P. M., Lehane, D., and Kulke, W.: Brit. M. l 1: 1486, 1961. 11. Clarke, C. A., Donnohoe, W. T. A., McConnell, R. B., Woodrow, l C., Finn, R., Krevans, l R., Kulke, W., Lehane, D., and Sheppard, P. M.: Brit. M. l 1: 979, 1963.