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Experimental production of anemia in fetal lambs
Experimental production of anemia in fetal lambs lAIN R. McFADYEN , M.B., M . R.C.O.G . · URABALA BOONYAPRAKOB , M.D . · · DONALD L . HUTCHI:'I
D E S PIT E excitin~ recent advances III diagnostic tcchniques, therapeutic procedures, and the introduction of preventivc measures, hemolytic disease of the newborn provides obstetricians and pediatricians with formidable problems. The fetuses at greatest risk continue to be those who develop severe anemia before reaching sufficient maturity to survive treatment by early induction of labor and exchange transfusion. Some fetuses with profound anemia develop hydrops fetal is, a clinically recognizable preterminal phase of the disease. Most physicians hold that the presence of hydrops is a contraindication to fetal transfusion, while a few prescribe this therapeutic measure with infrequent success under these circumstances. Elucidation of the pathophysiology of hydrops seems essential- if the condition is to be successfully treated with prevention of fetal death . This report represents an initial
attelllpt to produ(,t' an l'xperilll(' ntal model in the fetal lalllh which would simulate the physicorllt'lIlical and pathologic phenomena as scen in se\'cre human crythroblastosis. Thc hypothesis to be testl'd was that hydrops is the result of anemia and high output cardiac failure; an tHlsuccessful attempt to restore rcd blood cell Illass by increased erythropoiesis is followed by hypoxia, cardiac hypertrophy and dilatation, congestive failure, edema, anoxia, and death. The specific aim was to produce in a gradual controlled manner a severely anemic sheep fetus in order that we might determine when in the course of progressive anemia hydrops fetalis develops, how frequently it develops, and if at any stage it is reversible. The related clinical questions to be answered were the optimal time and frequency of fetal transfusion, the rate of blood administration to the fetus, and the optimal amount of blood needed to treat this condition. The therapeutic efficacy of other measures could be assessed if a reproducible model were obtained. No entirely satisfactory animal preparation was available to answer these questions. The sheep was selected as the animal of choice because of the availability of pregnant sheep of known gestational age, because of the relatively large size of the fetus, and because of our previous success in preventing premature Jabor after uterine surgery associated with
From the Departments of Obstetrics and Gvnecology and Pediatrics, University of Pittsburgh School of Medicine and Magee-Womens Hospital. This work was aided by General Research Support Grant FR-05570-02. Presented by invitation at the Seventyeighth Annual Meeting of the American Association of Obstetricians and Gynecologists, Hot Springs, Virginia, Sept. 7-9, 1967. ·Present address : St. Thomas's Hospital, London, S .E .l, England. ··Pediatric Research Fellow.
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the manipulation of the fetus and placement of chronic indwelling fetal catheters. Methods Pregnant hybrid sheep were admitted to the Animal Laboratory for observation, acclimatization, and base-line studies at approximately 110 days' gestation (normal gestation, approximately 150 days). Five hundred milligrams of progesterone was given intramuscularly the day prior to surgery and repeated daily thereafter for as long as the fetus remained alive. 1 Food was withheld for 18 hours before anesthesia was induced, and 5 per cent glucose in normal saline or Ringer's lactate solution was infused during the operation. Hysterotomy, amniotomy, and cannulization of the fetal jugular vein were carried out under halothane anesthesia at between 116 and 134 days of gestation. Heparinized normal saline was used to maintain catheter patency. In 2 instances, the fetus was removed from the uterus, weighed, and replaced without ill effect. On closing, warm isotonic saline containing penicillin and streptomycin was placed within the uterus in a volume approximating the lost amniotic fluid. The uterine incision was closed around the indwelling catheter which was then brought through the maternal abdominal wall and sutured to the skin. Antibiotics were initially given to only the mother postoperatively, but 5 of the first 8 fetal lambs died of infection; thereafter, 250 mg. of ampicillin was instilled through the fetal catheter daily and there was no further fetal infection. Fetal blood was withdrawn daily for measurement of the hematocrit and hemoglobin concentration. The pH, oxygen, and carbon
dioxide tensions and the base excess were estimated using the Radiometer pH meter, p02 electrode, and the Siggaard-Andersen nomogram. 2 Plasma proteins were measured with a refractometer, and a specimen of blood was cultured at frequent intervals. Results Initial efforts were directed toward the production of anemia and hydrops by gradual exsanguination of the fetus over a period of days. Three of the experiments in this group of 10 sheep were unsatisfactory due to difficulties with anesthesia, catheter blockage, and infection. Between 20 and 40 m!. of blood was removed daily from each fetus for 6 to 18 days (Table I). This amount constitutes approximately 5 to 10 per cent of the circulating blood volume. In all cases, the hemoglobin and hematocrit fell by 30 to 50 per cent. Both fetus and ewe tended to be acidotic during the surgical procedure; but thereafter, p02, pC0 2 , pH, base excess, and plasma proteins remained fairly constant. The results of fetal bleeding during 18 days in one lamb are shown in Fig. 1. The data from this fetus are typical of this group in that the hemoglobin and hematocrit decrease while the other analyses showed no consistent or significant change. Since hydrops fetal is had not been produced by gradual exsanguination to this degree, a second group of sheep was given phenylhydrazine hydrochloride intravenously in an effort to produce a predictable hemolytic anemia. The drug was first administered to 2 nonpregnant sheep. In both cases, significant anemia was produced without dis-
Table I. Anemia by bleeding Hematocrit
687
(%)
Hemoglobin (Gm.%)
Sheep No.
Days bled
Initial
Lowest
Initial
1 9 13 19 20 28 30
B 7 18 11 6 11 9
41 40 40 53 41 37 47
28 25 19 31 29 23 29
11.8 10.3 10.8 14.4 10.2 10.2 14.0
I
Lowest
B.O 5.8 5.2 B.5 7.7 6.3 B.2
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March I, 1968 & Gynec.
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EXSANGUINATION ,,-----,'-----,,----,,----~,----_,----_r----_r----T
/0 0,
"0....
/
0-0
".-.-., •"'-.•-." .. -. ............
•
20
'"
%
E
E
N
o
/o ....... o--()"""""""'
0 _ 0 __ 0-0..........
5
0- /0 0
°
...... /
/'
3
• / ".
<>.
40
20
125
127
129
131
DAYS
133
135
GESTATION
137
139
141
Fig. 1. Effects of 17 daily phlebotomies of from 20 to 40 m!. of fctal blood from Shcl'p No. 13.
PHENYLHYDRAZINE TO NON-PREGNANT SHEEP 60 50
.---.-. .--.------- '.
. .... ./
.040ml/ko
~
40
+
~ ...... -
.-.~.
" .......... '---1-·-,-·-· r".'"
......
~
+.02om./kO 0~0----2~--~4----~6--~8~--~10----1~2--~14----~16--~1-8---2~0~ DAYS
Fig. 2. Change in hematocrit after intravenous phenylhydrazine in 2 adult nonpregnant sheep.
cernible change in liver function tests (Fig. 2). Phenylhydrazine hydrochloride was then administered intravenously to 7 pregnant ewes in graduated doses between 0.125 and 0.75 gram per sheep. One of these ewes aborted a macerated 700 gram fetus 8 days after the drug was administered, and 3 ani-
mals represented unsatisfactory preparations because of technical difficulties. In all 4 of these ewes, however, the maternal hematocrit fell by between 30 and 50 per cent. In 2 of the 3 remaining satisfactory preparations, fetal anemia was also produced. The third fetus in this group developed hemoconcen-
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PHENYLHYDRAZINE TO MOTHER 5
3
20
'"
:J:
E
E
N
oQ.
o
/e-e-e ____ e_e-e '-
/e",
e
e-e
" - -___ L.nYlhYdrazin•.01 gm/kg
Jl9
121
123
125 DAYS
127
129
131
133
135
GESTATION
Fig. 3. Effect of maternally administered phenylhydrazine in fetal Lamb No.8.
Table II. Effect of phenylhydrazine on sheep fetus Sheep
Dose (mg.)
Fetal hematocrit (%) Lowest
Age
Initial
3
20
130
45
35
10
26 200
142 149
37 37
37 29
22
100 200
135 139
53 56
53 30·
*po. docre""od 63 per cont.
tration within 48 hours of the maternal injection with a simultaneous reduction in fetal blood volume of 50 per cent. This hemoconcentration persisted until the onset of labor on the fourteenth day after the injection of the drug (Fig. 3). The fetus presented as a breech and became impacted because of an abdominal tumor. During the manipulation and extraction of the lamb, there was a sudden release of a large amount of fluid followed by relatively easy delivery of a stillborn fetus. The fluid represented ascitic fluid which escaped from a rent found in the abdominal wall. There was also considerable clear fluid in the pleural cavity. This fetus may have been hydropic.
Finally, in 3 experiments, phenylhydrazine was given directly to the fetus through its indwelling catheter (Table II). Doses of 20 mg. produced no discernible response. Doses of 200 mg. were followed by a rapid fall in hematocrit in all cases which persisted for several days. Hemolysis was increased with repeated doses (Fig. 4). Two of these lambs died in utero and one was live born. This newborn lamb (Fig. 5) was lethargic, weak, and had respiratory distress for several hours immediately after delivery. Continued administration of phenylhydrazine in the neonatal period produced increasingly severe anemia; the lowest hematocrit was 14 per cent. Apart from the expected rise in pOa
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PHENYLHYDRAZINE
March I, 1968 II< Gynec.
J. Ob,!.
TO FETUS
20
so ~ I-
ii:
e
50
g
tiE 40
...2
0-.
\
•
•
e, e--e_e
135
137
139
DAYS
GESTATION
0.19m 0.29m phenylhydrazine
%:
0
___ e-e
~
'\e-e
30 129
131
133
141
143
145
147
Fig. 4. Change in hematocrit, pO" and prot .. in! in f('tal blood when phenylhydrazine is administered directly to the fetus. (Lamb No. 22.)
PHENYLHYDRAZINE TO FETUS 1-'
7
6
5
4
9"' phenylhydrazine
o 134
140
150
DAYS
10
20
30
Fig. 5. Change in hematocrit and proteins with continued administration of phenylhydrazine to newborn Lamb No. 10.
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and plasma proteins fol\owing delivery, there was no significant change in the other serum values which were llIeasured. Despite the anemia, the lamb showed no evidence of congestive cardiac failure.
Comment Failure to produce hydrops fetal is by chronic bleeding may have been due to our failure to achieve a sufficiently low hemoglobin or to our inability to maintain the anemia for a sufficient period of time. In the human, helllolysis usually continues for at least 12 weeks before the fetus becomes hydropic, and there is usually other evidence of tissue anoxia such as a reduction in plasma protein.~ In an attempt to prolong the period of anemia and to diminish the surgical trauma, other approaches were considered. The immunologic production of antibodies to sheep red cells was rejected because we possessed no special competency in this field and because of the suggestion by Cummings
REFERENCES
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and Bellville' that antibodies (at least to Salmonella pollorum) do not cross the placenta from mother to fetus. Antimitotic agents such as methotrexate and azathioprine were dismissed because of the likelihood of producing diffuse cellular damage in both fetus and ewe. 5 Phenylhydrazine administration was selected as a suitable method of inducing anemia because of the favorable results reported by Cruz 6 and others in producing hemolytic anemia in rats and rabbits. The erythrocytes are damaged and cleared from the circulation by the reticuloendothelial cells of spleen and liver, thus resembling the anemia of rhesus isoimmunization. The similarity is further enhanced by the reduction of oxygen-carrying capacity which accompanies this damage. The authors gratefully acknowledge the technical assistance of Mrs. Rosemary S. Fox, B.A., Mrs. Vera Waterman, B.S., and Mrs. Doris M. Watson, B.S.
I. Mcsehia, G., Cotler, j. R., Breathnach, C. S., and Barron, D. H.: Quart. J. Exper. Physio\.
4. Cummings, j. N., and Bellville, T. P.: AM. J. OasT. & GVNEC. 86: 504, 1963. 5. Rosenkrantz, J. G., Githens, J. H., Cox, S. M., and Kellum, D. L.: AM . J. OUST. & GVNEC. 97: 387, 1967. 6. Cruz, W.O.: Am. J. M. Se. 202: 781, 1941.
Discussion DR. CHARLES II. Ih:NllRICKS, Clt'vl'\and, Ohio. The hypothesis to he tested was "that hydrops is the resultant of anemia and high output cardiac failure." If the hypothesis were to be completely vindicated, this implies that high output cardiac failure should be associat('d with the anemia which they were going to induce. With one possible exception no hydrops was produced, and in no instance was there any evid('nce of high output cardiac failure. The authors are to be congratulated for conceiving and carrying through a difficult program of animal experill1l'ntatinn. It was probably inevitable that such a set of ('xperiments would be performed eventually in the shrep or in other
slIbhuman mammal species. It is very important that such negative results as those obtained in the current study should be carefully written up and published for three reasons: (A) so that no one will need to repeat the same work again, (B) because a carefully written negative report may increase ollr understanding of the pathophysiology of the subject under examination, and (C) because the development of new experimental techniques may prove useful to other workers. In their thinking the authors had equated hydrops with an advanced degree of erythroblastosis, which is a natural thing to do. Potterl points out, however, that hydrops is not by any means always due to erythroblastosis. It is not
50: 185, 1965. 2. Siggaard-Andersen, 0. : Scandinav. J. Clin. & Lab. Invest. 15: 211,1963. 3. Morison, j. E. : Focta1 and Nf'onatal Pathology, cd. 2, Washington, D. C., 1963, Butterworth & Company, Ltd., Chap. 11, p. 256.
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692 McFadyen, Boonyaprakob, and Hutchinson
uniformly associated with anemia or cardiac malformation, inasmuch as it can occur to the infants of women with toxemia, nephritis, and other chronic conditions. She notes that there is an associated deficiency of the plasma proteins, the values being at times as low as 1.5 to 2 Gill. per cent. She has noted evidence of a sharply diminished circulating blood volume.' These observations would in ad\'ance throw some doubt on the validity of the author's original hypothesis which equated hydrops with anemia plus high output cardiac failure in erythroblastosis. The authors fail to mention whether or not they searched for placental changes which might also resemble those seen in severe erythroblastosis. A recent study by Wentworth' has reconfirmed the classical findings that in severe hemolytic disease the placenta. is larger, paler, edematous, and more friable than the .normal placenta . Wentworth again raises the highly speculati\'e possibility that "the placental changes might he due directly to a reaction between the circulatory maternal antibodies and the D-positive cells rather than secondary to the fetal disease, i.e., hemolytic anemia and anoxia." He speculates further that "the pathologic changes in the hydropic fetus might also in part be a direct result of this reaction rather than just secondary to the hemolytic anemia."2 It should be pointed out that these experiments failed to produce another important facet of the syndrome of hydrops, the exceptionally low plasma protein level. Despite the anemia induced in these fetuses, the plasma protein levels tended to increase with time, and no more than a fraction of this increase can be accounted for on the basis of hemoconcentration. In the one fetus made anemic by phenylhydrazine in utero and who continued to receive this drug after delivery, the plasma protein levels, already adequate prior to delivery, rose subsequently to 6 Gm. per cent and were sustained there even though the fetus was constantly developing a more profound anemia . Incidentally, no cardiac failure could be detected in this newborn even when the hematocrit reached a level as low as 14 per cent. The authors state that their failure to induce hydrops may have been due to their failure to lower the fetal hemoglobin far enough (or a sufficient length of time to permit the complete syndrome to appear. This is undoubtedly an important point, since the lowest hematocrit achieved in utero was 19 per cent. A number of
Am.
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('J'ylhroblastotic hut nunhydrupic infants, deliwro'u with pl'J'iph"ral hl'lllatouits in the range of I:> to 18 pl'r cl'nt (and presumably with even lowI'r wnOllS hl'lllatonits ), ha\'e survived after appropriate c'xc'hangc~ transfusion therapy. One might ask why Dr. lIutdlinson and his coworkc'rs did not attl'mpt to lower the hematocrit lewis still further. Is it possible that, inasmuch as thl' fetal attrition was already high from the l'xllt'rilllc'ntal pn)('c'durc' alont', they did not beIi!'\'(, tht' fetus(,s could survive the added trauma of bringing the hematocrit down within the r,Hlgl' commonly ohserved in hydrops? Finally, while the sheep is a very useful expc·rimental model for studying the reproductive pro('es.~ in g"nl'ral, data dl'riVl'd from sheep studies arc not automatically translatable into hUlllan tC'rlllS or appli('ablc to IHunan pregnancy. Thlls, on the basis of sp"des difference alone, thnl' still r(,mains the possibility that the authors may be COffect in their original hypothesis. In \'il'w of the dinkal and pathologic observations whieh ha\'l' been made on this process, however, it appears likl'ly that the causation of the condition of hydrops is far more complex than the ml're presen,~ e of anemia and high output cardiac failure. REFERENCES
1. Potter, E. L.: Pathology of the Fetus and Infant, t~d. 2, Chicago, 1961, Year Book Medical Publishers. 2. Wentworth, P.: AM . J. OaST. & GVNEC. 98: 283, 1967.
DR. DANIEL G. MORTON, Los Angeles, California. Dr. Hutchinson's aim of devising a model for the elucidation of the pathophysiology of hydrops fetalis is indeed an important one jf we are to learn how to prevent this usually fatal condition. In approaching the problem it seems logical to me to test first the hypothesis that hydrops might simply be the result of progressive anemia and associated cardiac failure and this is what Dr. Hutchinson has attempted to do. Pregnant sheep between 116 and 134 days of gestation were chosen for the experiments. At hysterotomy a catheter was fixed in the jugular vein of the fetus and brought to the outside for subsequent bleeding and chemical observations. Between 20 and 40 m!. of blood was removed daily over a period of 6 to 18 days; p02' pC0 2 • pH, base excess, and plasma proteins were determined as well as hemoglobin and hematocrit values. As related by the author, the hemoglobin and hematocrit decreased 30 to 50 per cent, but
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the other analyses showed no consistent changes and there was no evidence of hydrops. Having failed in these efforts, phenylhydrazine was used to produce hemolytic anemia in the ewes and also in the fetuses; this was efTective in thc production of anpl11ia in the fetLises but not in the production of hydrops, with one possible exception. In regard to ancmia in the lamb fetus, Dr. Thomas Kirsrhbaum of our staff called to my attention an observation made by Barcroft 1 in his book, Researches on Prenatal Life, published 20 years ago: "Experiment 17. 126 days, twins. Left foetlls observed. Oxygl'n Capacity 11.7. Percentage saturation in umhilical artery 46. The right fetus had been dead in the uterus for some days and was quite oedpmatous and unfit for weighing and measuring." Hc furtl1('r added that thc observation was suggestive of the possihility of a fctus becoming anemic and that such an anemia might bc attributed to more than one causc, one of which was "some st' rological phenomenon comparable to the Rh factor." In 1958, Cummings and Kaiser 3 published observations on spontaneously occurring anemia in 4 fetal lambs in 3 prcgnancies of 87, !07,and 126 days' gestation, respectively. Their findings compared closely with the norms which these same authors had obtained in previous studies except of course for the low hemoglobins, which ranged from 4.5 to 9.4 Gm. Plasma proteins were normal as werc the liver and spleen weights. They observed that the findings were unlike the changes associated with severe erythroblastosis. I would also like to call to your attention a study of induced am'mia in fctal monkeys reported by Karlis Adamsons f (in the Transcript of the Third Roehl'ster Trophoblast Conference, November, 1965. Editrd by Lund and Thiede); the bleedings were carried to the point of finally producing the death of thc fetllses. While signs of heart failure appeared eventually the typical features of hydrops were absent. It appears that so far something of importance in the production of hydrops is being missed. While in all probability the missing factor is much more fundamental than an earlier and more gradually developing fetal anemia than that produced in Hutchinson's experiments, I do think that this point needs to be considered. In all probability blood dClitruction in the fetus begins at an early period of gestation in severe
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erythroblastosis in the human and continues at an unknown and possibly varying r:::e. When the severe manifestation of hydrops develops there is always marked extramedullary erythropoiesis and marked hepatomegaly and splenomegaly. I would like to ask Dr. Hutchinson if blood smears were examined for erythroblasts, nucleated red cells, etc., and if microscopic sections of the livers were studied? Occasionally, severe and fatal anemia of nonhemolytic type has been recognized in the newborn in which there was no edema, no jaundice, and no hepatomegaly. On the other hand, hydrops has been encountered occasionally without hemolytic anemia. It may be that an immunologic mechanism will have to be invoked before hydrops of the type we are looking for can be produced. It may be that the causative mechanism is one that not only produces blood destruction and its conse~ quences but also damages primarily certain other tjssues such as the liver; and spleen. One can only speculate but it is highly probable that a different approach will have to be taken. REFERENCES
1. Barcroft, J.: Researches on Prenatal Life, Baltimore, 1947, The Williams & Wilkins Company. . 2. Cummings, J. N., and Kaiser, .I. H.: ttudes neonatal. VII: 127, 1958. 3. Kaiser, I. H., and Cummings, J. N.: J. AppJ. Phy!iol. 10: 484, 1957. 4. Adamson!, K.: Acid-Base State and Cardiovascular Respome During Induced Anemia in Utero in the Rhesus Monkey, Transcript of the Third Rochester Trophoblast Conference, 1965, p. 129.
DR. JOHN S. ZELENIK,* Chiang Mai, Thailand. To one who has been interested in the problem of erythroblastosis for most of his professional life, the title of Dr. Hutchinson's paper was particularly interesting and exciting. It was disappointing to hear that he was not able to produce hydrops. The disappointment was more profound since the techniques employed to develop the anemia seemed to be reasonable ones. I would like to take issue with the preceding discussants, however, in that I believe that anemia is still an essential and probably the most esseritial component in etiology of hydrops. In our university hospital at Chiang Mai in northern Thailand we see a case of hydrops on the average of once a month. Of the last 33 hydropic infants *By invitat.ion.
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McFadyen, Boonyaprakob, and Hutchinson
that wt'rt' delivered th ere in the past J ycars, none W;lS the result of Rh or other blood incompatibilities, for ll'ss than I pt'r n'nt of thl' Tha i's arc Rh negative. Almost all wcre the rrsult of a h(,lllogloilinopathy duc to what is known as llan's hemoglobin. This abt'rrant IWllloglohin is genetically transmitted through both sext's. Whrn the homozygous state dcvl'iops, a disease Offurs within the fetus that results in an anemia of such severity that none of thcse homozygolls fetuses survivc. Most die in utero bctw<,en thl' thirtieth and thirty-fourth week, and all of thl'llI arc hydropic. In addition to the lIIll'lllia, t hl' edema and ascit('s, they all hav(' low total Sl'rllm proteins. The gross and microscopic features of the placenta and the tissues of the ktus an' exactly th e same as those found in hydrops of erythroblastosis fetal is associated with Rh incompatibil ity. These infants with Bart's hemoglobin, however, have no antibodies to their fl'd blood ('('I/s. Thrir's is a hrmoglobin prohl,'m in which the hrmoglobin is ineffirirnt, is una hie to handle its iron, and brings about a typl' of .. han~(' in the red blood cells, resulting in th('ir dl'stru('tion in the spleen at an ('verinc.-rasing ratl'. Thus anoxia is the result of inefficient hinding with hemoglobin and with drcr('asing numbers of red blood cells. I would like to ask Dr. Hutchinson thc same questions that have been presented previously: What did the livers and spleens show? What did the studies on his placentas show? A few years ago Dr, Alvarez in Montevideo suggested that one could diagnose and prognosticate the outcome of erythroblastotic infants in utero by needle biopsies of the placenta. We actually tried a couple of cases, but fortunately Dr. Liley's work appeared at the same time, so we pnrsul'd his simpll'r method of amniocentl'sis for this diaRnosis and prognostication. Alvarl'z, using phase microscopy to study hi~ biopsy material, pointed out that with p:-ogressi\'t' erythroblastosis there was a histolog:c ch ~ ng(' of the placenta to the immature state, that mark('d ~yncytial proliferation occurrrd, and that nl'W syncytial sprouts developrd. We studied sevl'ral of the placentas from our fetusl's that had hydrops as a rl'sult of Bart's hrmoglobin, both undrr phase microscopy and using vital staining, and found that thl'y apprarl'd exartly like the hiopsil's from sevt'rely affrctt'd infants wht're tht' erythroblastosis and / or hydrops was due to Rh incompatibility. In Bart's h('moglobinopathy the anemia is not related to blood destruction by anti-
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hody but it is related to hlood destruction. AI. though the hlood d .. strut:tioll is g<'netically engl'nd.' red th., anemias are just as Sl'vere and the pathol"KY is the , .. me in both instances. I alii II lit as p"ssilllistit: as thl' previous two dis(,lIssants ;lIId I do hope that Dr. Hutchinson has not hl'l'n dis('I)\II'a~l'd to the point of discontinuing th(' study. I fl'rl that he has not been SlllTl'ssful IWl'aUSl' hl' has nut dl'\'e\opl'd a severe .'nollgh dl'gfl'l' of anemia in his experimental 'lIIimals to bring ahollt a tl'llr erythroblastic I"l'SpOnsl'. I furthl'!, fel'! that hydrops is probably a catadpllIil' tyPt' of t'n'nt which Occurs in hours or prohably not longt'f than a period DE days. Tht'fl' was onl' most int,'resting finding as far as I was conccrned, and do not know whether it impn'sS/'d him or not , and that was the fact that in all of his f"t,,1 lamhs the total serum protein was risinl!;. Could this Ql' a mechanism to replat't' t hI' hlood \'0111111<' lost? If he had persisted, how,'\'('r. in innl'asing thl' anemia in his fetuses, I helil'\'r that this prott'in would have rl'wrsl'd itself as proll;fl'ssi\'l' anemia brgan to aft"l't th., liver ,lI1d inted.'re with protein synthesis and as progn'ssiw anoxia affected the heart and thl' vascular Ill'd, osmotic prrssure changes in the cirnllating systl'm must occur which then result in the gen{'ralizt'd hydrops. We get an erythroblastosis-like picture in other conditions such as syphilis and diabetes, Perhaps here there are changes that occur within the placental vasculature 50 that there is a decrease in, and interference with, the transfer of oxygen, The placenta compensates by enlarging, the fetal erythropoetic syst('ms respond with more cells but as the oxyg('n tension continues to fall t~ leVl'ls that arl' {'wntually damaging to vital struc. IIlI'rs, su('h as the liwr, the hrart, and the vas. ('ular Iwd, dl'gl'nl'rati\'e changes occur that reo suit in hydrops. I pr('di('t that if Dr. Hutchinson's \iv('r or bone marrow studies show any erythropoetic response, ht' will he ahle to produce hydrops jf he will continue to do the hleedinR at a progressive rate rather than at a constant rate. This is undoubtedly the mechanism in thl" casl's of blood incompatibility. Certainly his hl'moglobin and hemato. crit Il'wls must 1)(' dl'opPt'd to nllt<'h lower levels OWl' mu('h longer P<'riod~ of time. DR. HCTCHlN!'ON (Closing) . It was not my {'x(J<'ctation at th .. outsrt of this study that the results would br so totally negative. It may very well be that the right question ha! not been asked, The finding! associated with hydrops may
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nOI be set:oll(Jary to anl'mia but may, in facl, represent an imlllulle respollse to maternal antigen. Gross and Illicroscopit: observation of plat:ental cot yledons has not l)('en very rl'vl'alillg. It is difficult 10 make comparative statements, bllt there was no histologic evidence of villus edema or of proliferalion of trophoblast or stroma. All lambs which wt'fe not macerated were autopsied. No significant differences were found in tIll' weight or appearance of fetal organs. Precis/' quantitative morphologic studit's were not dOlle. It does set'm possible that hypoproteinemia may be a very important factor in the development of anasarca and Sl'rous effusions. We considered thl' possibility of trying to suppress protein synthesis in the ft,tus amI plat·t'nta but rl'jected it hecause of our concern that we might produce diffuse ct'lllllar changes with the chemicals which might make it difficult to dt·termine the specific calise of any ohsl'rvl'd changl'. I really do nOI know how long onl' could maintain a significant hypoprotl'ilwmic statl' in the fetus. Ndtht'r do I know what tht' threshold of anemia is below which hydrops fetalis will consistently develop. Mr. Roul'lll' told me recently that he had observed Ihe characteristic features
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of hydrops wilh a fetal hemoglobin of 80 pel' cc'n!. I believe that in general the more severe the anemia, the more profound the tissue hypoxia and the more likely that there will be free fluid or massive edema. It is my understanding that the monkey expl'riments of Adamsons were very acute studies. We thought that we might be successful by prolonging the fetal bleeding with this more chronic approach. The hydrops associated with abnormal hemoglobins such as Bart's is most interesting. Apparently tissue hypoxia occurs at relatively high hemoglobin levels because the abnormal hemoglobin does not release oxygen properly. A possible mechanism of action of phenylhydrazine is Ihe production of some change in hemoglobin which will decrl'ase its oxygen-carrying capacity. A variety of conditions have been associated wilh hydrops ft,talis. Syphilis and diabetes might slIggpst a matPrnal influence. Fetal-fetal transfllsions and fetal-maternal bleeds suggest that fptal anl'mia may cause the syndrome. The hydrops associalC'd with erythroblastosis and with abnormal hemoglobin levels may well suggest some primary basic immune mechanism.
D E S PIT E excitin~ recent advances III diagnostic tcchniques, therapeutic procedures, and the introduction of preventivc measures, hemolytic disease of the newborn provides obstetricians and pediatricians with formidable problems. The fetuses at greatest risk continue to be those who develop severe anemia before reaching sufficient maturity to survive treatment by early induction of labor and exchange transfusion. Some fetuses with profound anemia develop hydrops fetal is, a clinically recognizable preterminal phase of the disease. Most physicians hold that the presence of hydrops is a contraindication to fetal transfusion, while a few prescribe this therapeutic measure with infrequent success under these circumstances. Elucidation of the pathophysiology of hydrops seems essential- if the condition is to be successfully treated with prevention of fetal death . This report represents an initial
attelllpt to produ(,t' an l'xperilll(' ntal model in the fetal lalllh which would simulate the physicorllt'lIlical and pathologic phenomena as scen in se\'cre human crythroblastosis. Thc hypothesis to be testl'd was that hydrops is the result of anemia and high output cardiac failure; an tHlsuccessful attempt to restore rcd blood cell Illass by increased erythropoiesis is followed by hypoxia, cardiac hypertrophy and dilatation, congestive failure, edema, anoxia, and death. The specific aim was to produce in a gradual controlled manner a severely anemic sheep fetus in order that we might determine when in the course of progressive anemia hydrops fetalis develops, how frequently it develops, and if at any stage it is reversible. The related clinical questions to be answered were the optimal time and frequency of fetal transfusion, the rate of blood administration to the fetus, and the optimal amount of blood needed to treat this condition. The therapeutic efficacy of other measures could be assessed if a reproducible model were obtained. No entirely satisfactory animal preparation was available to answer these questions. The sheep was selected as the animal of choice because of the availability of pregnant sheep of known gestational age, because of the relatively large size of the fetus, and because of our previous success in preventing premature Jabor after uterine surgery associated with
From the Departments of Obstetrics and Gvnecology and Pediatrics, University of Pittsburgh School of Medicine and Magee-Womens Hospital. This work was aided by General Research Support Grant FR-05570-02. Presented by invitation at the Seventyeighth Annual Meeting of the American Association of Obstetricians and Gynecologists, Hot Springs, Virginia, Sept. 7-9, 1967. ·Present address : St. Thomas's Hospital, London, S .E .l, England. ··Pediatric Research Fellow.
686
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Experimental production of toxemia
the manipulation of the fetus and placement of chronic indwelling fetal catheters. Methods Pregnant hybrid sheep were admitted to the Animal Laboratory for observation, acclimatization, and base-line studies at approximately 110 days' gestation (normal gestation, approximately 150 days). Five hundred milligrams of progesterone was given intramuscularly the day prior to surgery and repeated daily thereafter for as long as the fetus remained alive. 1 Food was withheld for 18 hours before anesthesia was induced, and 5 per cent glucose in normal saline or Ringer's lactate solution was infused during the operation. Hysterotomy, amniotomy, and cannulization of the fetal jugular vein were carried out under halothane anesthesia at between 116 and 134 days of gestation. Heparinized normal saline was used to maintain catheter patency. In 2 instances, the fetus was removed from the uterus, weighed, and replaced without ill effect. On closing, warm isotonic saline containing penicillin and streptomycin was placed within the uterus in a volume approximating the lost amniotic fluid. The uterine incision was closed around the indwelling catheter which was then brought through the maternal abdominal wall and sutured to the skin. Antibiotics were initially given to only the mother postoperatively, but 5 of the first 8 fetal lambs died of infection; thereafter, 250 mg. of ampicillin was instilled through the fetal catheter daily and there was no further fetal infection. Fetal blood was withdrawn daily for measurement of the hematocrit and hemoglobin concentration. The pH, oxygen, and carbon
dioxide tensions and the base excess were estimated using the Radiometer pH meter, p02 electrode, and the Siggaard-Andersen nomogram. 2 Plasma proteins were measured with a refractometer, and a specimen of blood was cultured at frequent intervals. Results Initial efforts were directed toward the production of anemia and hydrops by gradual exsanguination of the fetus over a period of days. Three of the experiments in this group of 10 sheep were unsatisfactory due to difficulties with anesthesia, catheter blockage, and infection. Between 20 and 40 m!. of blood was removed daily from each fetus for 6 to 18 days (Table I). This amount constitutes approximately 5 to 10 per cent of the circulating blood volume. In all cases, the hemoglobin and hematocrit fell by 30 to 50 per cent. Both fetus and ewe tended to be acidotic during the surgical procedure; but thereafter, p02, pC0 2 , pH, base excess, and plasma proteins remained fairly constant. The results of fetal bleeding during 18 days in one lamb are shown in Fig. 1. The data from this fetus are typical of this group in that the hemoglobin and hematocrit decrease while the other analyses showed no consistent or significant change. Since hydrops fetal is had not been produced by gradual exsanguination to this degree, a second group of sheep was given phenylhydrazine hydrochloride intravenously in an effort to produce a predictable hemolytic anemia. The drug was first administered to 2 nonpregnant sheep. In both cases, significant anemia was produced without dis-
Table I. Anemia by bleeding Hematocrit
687
(%)
Hemoglobin (Gm.%)
Sheep No.
Days bled
Initial
Lowest
Initial
1 9 13 19 20 28 30
B 7 18 11 6 11 9
41 40 40 53 41 37 47
28 25 19 31 29 23 29
11.8 10.3 10.8 14.4 10.2 10.2 14.0
I
Lowest
B.O 5.8 5.2 B.5 7.7 6.3 B.2
688
McFadyen, Boonyaprakob, and Hutchinson
Am.
March I, 1968 & Gynec.
J. Ob,!.
EXSANGUINATION ,,-----,'-----,,----,,----~,----_,----_r----_r----T
/0 0,
"0....
/
0-0
".-.-., •"'-.•-." .. -. ............
•
20
'"
%
E
E
N
o
/o ....... o--()"""""""'
0 _ 0 __ 0-0..........
5
0- /0 0
°
...... /
/'
3
• / ".
<>.
40
20
125
127
129
131
DAYS
133
135
GESTATION
137
139
141
Fig. 1. Effects of 17 daily phlebotomies of from 20 to 40 m!. of fctal blood from Shcl'p No. 13.
PHENYLHYDRAZINE TO NON-PREGNANT SHEEP 60 50
.---.-. .--.------- '.
. .... ./
.040ml/ko
~
40
+
~ ...... -
.-.~.
" .......... '---1-·-,-·-· r".'"
......
~
+.02om./kO 0~0----2~--~4----~6--~8~--~10----1~2--~14----~16--~1-8---2~0~ DAYS
Fig. 2. Change in hematocrit after intravenous phenylhydrazine in 2 adult nonpregnant sheep.
cernible change in liver function tests (Fig. 2). Phenylhydrazine hydrochloride was then administered intravenously to 7 pregnant ewes in graduated doses between 0.125 and 0.75 gram per sheep. One of these ewes aborted a macerated 700 gram fetus 8 days after the drug was administered, and 3 ani-
mals represented unsatisfactory preparations because of technical difficulties. In all 4 of these ewes, however, the maternal hematocrit fell by between 30 and 50 per cent. In 2 of the 3 remaining satisfactory preparations, fetal anemia was also produced. The third fetus in this group developed hemoconcen-
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Experimental production of toxemia
689
PHENYLHYDRAZINE TO MOTHER 5
3
20
'"
:J:
E
E
N
oQ.
o
/e-e-e ____ e_e-e '-
/e",
e
e-e
" - -___ L.nYlhYdrazin•.01 gm/kg
Jl9
121
123
125 DAYS
127
129
131
133
135
GESTATION
Fig. 3. Effect of maternally administered phenylhydrazine in fetal Lamb No.8.
Table II. Effect of phenylhydrazine on sheep fetus Sheep
Dose (mg.)
Fetal hematocrit (%) Lowest
Age
Initial
3
20
130
45
35
10
26 200
142 149
37 37
37 29
22
100 200
135 139
53 56
53 30·
*po. docre""od 63 per cont.
tration within 48 hours of the maternal injection with a simultaneous reduction in fetal blood volume of 50 per cent. This hemoconcentration persisted until the onset of labor on the fourteenth day after the injection of the drug (Fig. 3). The fetus presented as a breech and became impacted because of an abdominal tumor. During the manipulation and extraction of the lamb, there was a sudden release of a large amount of fluid followed by relatively easy delivery of a stillborn fetus. The fluid represented ascitic fluid which escaped from a rent found in the abdominal wall. There was also considerable clear fluid in the pleural cavity. This fetus may have been hydropic.
Finally, in 3 experiments, phenylhydrazine was given directly to the fetus through its indwelling catheter (Table II). Doses of 20 mg. produced no discernible response. Doses of 200 mg. were followed by a rapid fall in hematocrit in all cases which persisted for several days. Hemolysis was increased with repeated doses (Fig. 4). Two of these lambs died in utero and one was live born. This newborn lamb (Fig. 5) was lethargic, weak, and had respiratory distress for several hours immediately after delivery. Continued administration of phenylhydrazine in the neonatal period produced increasingly severe anemia; the lowest hematocrit was 14 per cent. Apart from the expected rise in pOa
690
McFadyen, Boonyaprokob, and Hutchinson
Am.
PHENYLHYDRAZINE
March I, 1968 II< Gynec.
J. Ob,!.
TO FETUS
20
so ~ I-
ii:
e
50
g
tiE 40
...2
0-.
\
•
•
e, e--e_e
135
137
139
DAYS
GESTATION
0.19m 0.29m phenylhydrazine
%:
0
___ e-e
~
'\e-e
30 129
131
133
141
143
145
147
Fig. 4. Change in hematocrit, pO" and prot .. in! in f('tal blood when phenylhydrazine is administered directly to the fetus. (Lamb No. 22.)
PHENYLHYDRAZINE TO FETUS 1-'
7
6
5
4
9"' phenylhydrazine
o 134
140
150
DAYS
10
20
30
Fig. 5. Change in hematocrit and proteins with continued administration of phenylhydrazine to newborn Lamb No. 10.
Volume 100 Number 5
and plasma proteins fol\owing delivery, there was no significant change in the other serum values which were llIeasured. Despite the anemia, the lamb showed no evidence of congestive cardiac failure.
Comment Failure to produce hydrops fetal is by chronic bleeding may have been due to our failure to achieve a sufficiently low hemoglobin or to our inability to maintain the anemia for a sufficient period of time. In the human, helllolysis usually continues for at least 12 weeks before the fetus becomes hydropic, and there is usually other evidence of tissue anoxia such as a reduction in plasma protein.~ In an attempt to prolong the period of anemia and to diminish the surgical trauma, other approaches were considered. The immunologic production of antibodies to sheep red cells was rejected because we possessed no special competency in this field and because of the suggestion by Cummings
REFERENCES
Experimental production of toxemia
691
and Bellville' that antibodies (at least to Salmonella pollorum) do not cross the placenta from mother to fetus. Antimitotic agents such as methotrexate and azathioprine were dismissed because of the likelihood of producing diffuse cellular damage in both fetus and ewe. 5 Phenylhydrazine administration was selected as a suitable method of inducing anemia because of the favorable results reported by Cruz 6 and others in producing hemolytic anemia in rats and rabbits. The erythrocytes are damaged and cleared from the circulation by the reticuloendothelial cells of spleen and liver, thus resembling the anemia of rhesus isoimmunization. The similarity is further enhanced by the reduction of oxygen-carrying capacity which accompanies this damage. The authors gratefully acknowledge the technical assistance of Mrs. Rosemary S. Fox, B.A., Mrs. Vera Waterman, B.S., and Mrs. Doris M. Watson, B.S.
I. Mcsehia, G., Cotler, j. R., Breathnach, C. S., and Barron, D. H.: Quart. J. Exper. Physio\.
4. Cummings, j. N., and Bellville, T. P.: AM. J. OasT. & GVNEC. 86: 504, 1963. 5. Rosenkrantz, J. G., Githens, J. H., Cox, S. M., and Kellum, D. L.: AM . J. OUST. & GVNEC. 97: 387, 1967. 6. Cruz, W.O.: Am. J. M. Se. 202: 781, 1941.
Discussion DR. CHARLES II. Ih:NllRICKS, Clt'vl'\and, Ohio. The hypothesis to he tested was "that hydrops is the resultant of anemia and high output cardiac failure." If the hypothesis were to be completely vindicated, this implies that high output cardiac failure should be associat('d with the anemia which they were going to induce. With one possible exception no hydrops was produced, and in no instance was there any evid('nce of high output cardiac failure. The authors are to be congratulated for conceiving and carrying through a difficult program of animal experill1l'ntatinn. It was probably inevitable that such a set of ('xperiments would be performed eventually in the shrep or in other
slIbhuman mammal species. It is very important that such negative results as those obtained in the current study should be carefully written up and published for three reasons: (A) so that no one will need to repeat the same work again, (B) because a carefully written negative report may increase ollr understanding of the pathophysiology of the subject under examination, and (C) because the development of new experimental techniques may prove useful to other workers. In their thinking the authors had equated hydrops with an advanced degree of erythroblastosis, which is a natural thing to do. Potterl points out, however, that hydrops is not by any means always due to erythroblastosis. It is not
50: 185, 1965. 2. Siggaard-Andersen, 0. : Scandinav. J. Clin. & Lab. Invest. 15: 211,1963. 3. Morison, j. E. : Focta1 and Nf'onatal Pathology, cd. 2, Washington, D. C., 1963, Butterworth & Company, Ltd., Chap. 11, p. 256.
March I, 1968
692 McFadyen, Boonyaprakob, and Hutchinson
uniformly associated with anemia or cardiac malformation, inasmuch as it can occur to the infants of women with toxemia, nephritis, and other chronic conditions. She notes that there is an associated deficiency of the plasma proteins, the values being at times as low as 1.5 to 2 Gill. per cent. She has noted evidence of a sharply diminished circulating blood volume.' These observations would in ad\'ance throw some doubt on the validity of the author's original hypothesis which equated hydrops with anemia plus high output cardiac failure in erythroblastosis. The authors fail to mention whether or not they searched for placental changes which might also resemble those seen in severe erythroblastosis. A recent study by Wentworth' has reconfirmed the classical findings that in severe hemolytic disease the placenta. is larger, paler, edematous, and more friable than the .normal placenta . Wentworth again raises the highly speculati\'e possibility that "the placental changes might he due directly to a reaction between the circulatory maternal antibodies and the D-positive cells rather than secondary to the fetal disease, i.e., hemolytic anemia and anoxia." He speculates further that "the pathologic changes in the hydropic fetus might also in part be a direct result of this reaction rather than just secondary to the hemolytic anemia."2 It should be pointed out that these experiments failed to produce another important facet of the syndrome of hydrops, the exceptionally low plasma protein level. Despite the anemia induced in these fetuses, the plasma protein levels tended to increase with time, and no more than a fraction of this increase can be accounted for on the basis of hemoconcentration. In the one fetus made anemic by phenylhydrazine in utero and who continued to receive this drug after delivery, the plasma protein levels, already adequate prior to delivery, rose subsequently to 6 Gm. per cent and were sustained there even though the fetus was constantly developing a more profound anemia . Incidentally, no cardiac failure could be detected in this newborn even when the hematocrit reached a level as low as 14 per cent. The authors state that their failure to induce hydrops may have been due to their failure to lower the fetal hemoglobin far enough (or a sufficient length of time to permit the complete syndrome to appear. This is undoubtedly an important point, since the lowest hematocrit achieved in utero was 19 per cent. A number of
Am.
J. Obst. &: Gynec.
('J'ylhroblastotic hut nunhydrupic infants, deliwro'u with pl'J'iph"ral hl'lllatouits in the range of I:> to 18 pl'r cl'nt (and presumably with even lowI'r wnOllS hl'lllatonits ), ha\'e survived after appropriate c'xc'hangc~ transfusion therapy. One might ask why Dr. lIutdlinson and his coworkc'rs did not attl'mpt to lower the hematocrit lewis still further. Is it possible that, inasmuch as thl' fetal attrition was already high from the l'xllt'rilllc'ntal pn)('c'durc' alont', they did not beIi!'\'(, tht' fetus(,s could survive the added trauma of bringing the hematocrit down within the r,Hlgl' commonly ohserved in hydrops? Finally, while the sheep is a very useful expc·rimental model for studying the reproductive pro('es.~ in g"nl'ral, data dl'riVl'd from sheep studies arc not automatically translatable into hUlllan tC'rlllS or appli('ablc to IHunan pregnancy. Thlls, on the basis of sp"des difference alone, thnl' still r(,mains the possibility that the authors may be COffect in their original hypothesis. In \'il'w of the dinkal and pathologic observations whieh ha\'l' been made on this process, however, it appears likl'ly that the causation of the condition of hydrops is far more complex than the ml're presen,~ e of anemia and high output cardiac failure. REFERENCES
1. Potter, E. L.: Pathology of the Fetus and Infant, t~d. 2, Chicago, 1961, Year Book Medical Publishers. 2. Wentworth, P.: AM . J. OaST. & GVNEC. 98: 283, 1967.
DR. DANIEL G. MORTON, Los Angeles, California. Dr. Hutchinson's aim of devising a model for the elucidation of the pathophysiology of hydrops fetalis is indeed an important one jf we are to learn how to prevent this usually fatal condition. In approaching the problem it seems logical to me to test first the hypothesis that hydrops might simply be the result of progressive anemia and associated cardiac failure and this is what Dr. Hutchinson has attempted to do. Pregnant sheep between 116 and 134 days of gestation were chosen for the experiments. At hysterotomy a catheter was fixed in the jugular vein of the fetus and brought to the outside for subsequent bleeding and chemical observations. Between 20 and 40 m!. of blood was removed daily over a period of 6 to 18 days; p02' pC0 2 • pH, base excess, and plasma proteins were determined as well as hemoglobin and hematocrit values. As related by the author, the hemoglobin and hematocrit decreased 30 to 50 per cent, but
Volume 100 Number 5
the other analyses showed no consistent changes and there was no evidence of hydrops. Having failed in these efforts, phenylhydrazine was used to produce hemolytic anemia in the ewes and also in the fetuses; this was efTective in thc production of anpl11ia in the fetLises but not in the production of hydrops, with one possible exception. In regard to ancmia in the lamb fetus, Dr. Thomas Kirsrhbaum of our staff called to my attention an observation made by Barcroft 1 in his book, Researches on Prenatal Life, published 20 years ago: "Experiment 17. 126 days, twins. Left foetlls observed. Oxygl'n Capacity 11.7. Percentage saturation in umhilical artery 46. The right fetus had been dead in the uterus for some days and was quite oedpmatous and unfit for weighing and measuring." Hc furtl1('r added that thc observation was suggestive of the possihility of a fctus becoming anemic and that such an anemia might bc attributed to more than one causc, one of which was "some st' rological phenomenon comparable to the Rh factor." In 1958, Cummings and Kaiser 3 published observations on spontaneously occurring anemia in 4 fetal lambs in 3 prcgnancies of 87, !07,and 126 days' gestation, respectively. Their findings compared closely with the norms which these same authors had obtained in previous studies except of course for the low hemoglobins, which ranged from 4.5 to 9.4 Gm. Plasma proteins were normal as werc the liver and spleen weights. They observed that the findings were unlike the changes associated with severe erythroblastosis. I would also like to call to your attention a study of induced am'mia in fctal monkeys reported by Karlis Adamsons f (in the Transcript of the Third Roehl'ster Trophoblast Conference, November, 1965. Editrd by Lund and Thiede); the bleedings were carried to the point of finally producing the death of thc fetllses. While signs of heart failure appeared eventually the typical features of hydrops were absent. It appears that so far something of importance in the production of hydrops is being missed. While in all probability the missing factor is much more fundamental than an earlier and more gradually developing fetal anemia than that produced in Hutchinson's experiments, I do think that this point needs to be considered. In all probability blood dClitruction in the fetus begins at an early period of gestation in severe
Experimental production of toxemia
693
erythroblastosis in the human and continues at an unknown and possibly varying r:::e. When the severe manifestation of hydrops develops there is always marked extramedullary erythropoiesis and marked hepatomegaly and splenomegaly. I would like to ask Dr. Hutchinson if blood smears were examined for erythroblasts, nucleated red cells, etc., and if microscopic sections of the livers were studied? Occasionally, severe and fatal anemia of nonhemolytic type has been recognized in the newborn in which there was no edema, no jaundice, and no hepatomegaly. On the other hand, hydrops has been encountered occasionally without hemolytic anemia. It may be that an immunologic mechanism will have to be invoked before hydrops of the type we are looking for can be produced. It may be that the causative mechanism is one that not only produces blood destruction and its conse~ quences but also damages primarily certain other tjssues such as the liver; and spleen. One can only speculate but it is highly probable that a different approach will have to be taken. REFERENCES
1. Barcroft, J.: Researches on Prenatal Life, Baltimore, 1947, The Williams & Wilkins Company. . 2. Cummings, J. N., and Kaiser, .I. H.: ttudes neonatal. VII: 127, 1958. 3. Kaiser, I. H., and Cummings, J. N.: J. AppJ. Phy!iol. 10: 484, 1957. 4. Adamson!, K.: Acid-Base State and Cardiovascular Respome During Induced Anemia in Utero in the Rhesus Monkey, Transcript of the Third Rochester Trophoblast Conference, 1965, p. 129.
DR. JOHN S. ZELENIK,* Chiang Mai, Thailand. To one who has been interested in the problem of erythroblastosis for most of his professional life, the title of Dr. Hutchinson's paper was particularly interesting and exciting. It was disappointing to hear that he was not able to produce hydrops. The disappointment was more profound since the techniques employed to develop the anemia seemed to be reasonable ones. I would like to take issue with the preceding discussants, however, in that I believe that anemia is still an essential and probably the most esseritial component in etiology of hydrops. In our university hospital at Chiang Mai in northern Thailand we see a case of hydrops on the average of once a month. Of the last 33 hydropic infants *By invitat.ion.
694
McFadyen, Boonyaprakob, and Hutchinson
that wt'rt' delivered th ere in the past J ycars, none W;lS the result of Rh or other blood incompatibilities, for ll'ss than I pt'r n'nt of thl' Tha i's arc Rh negative. Almost all wcre the rrsult of a h(,lllogloilinopathy duc to what is known as llan's hemoglobin. This abt'rrant IWllloglohin is genetically transmitted through both sext's. Whrn the homozygous state dcvl'iops, a disease Offurs within the fetus that results in an anemia of such severity that none of thcse homozygolls fetuses survivc. Most die in utero bctw<,en thl' thirtieth and thirty-fourth week, and all of thl'llI arc hydropic. In addition to the lIIll'lllia, t hl' edema and ascit('s, they all hav(' low total Sl'rllm proteins. The gross and microscopic features of the placenta and the tissues of the ktus an' exactly th e same as those found in hydrops of erythroblastosis fetal is associated with Rh incompatibil ity. These infants with Bart's hemoglobin, however, have no antibodies to their fl'd blood ('('I/s. Thrir's is a hrmoglobin prohl,'m in which the hrmoglobin is ineffirirnt, is una hie to handle its iron, and brings about a typl' of .. han~(' in the red blood cells, resulting in th('ir dl'stru('tion in the spleen at an ('verinc.-rasing ratl'. Thus anoxia is the result of inefficient hinding with hemoglobin and with drcr('asing numbers of red blood cells. I would like to ask Dr. Hutchinson thc same questions that have been presented previously: What did the livers and spleens show? What did the studies on his placentas show? A few years ago Dr, Alvarez in Montevideo suggested that one could diagnose and prognosticate the outcome of erythroblastotic infants in utero by needle biopsies of the placenta. We actually tried a couple of cases, but fortunately Dr. Liley's work appeared at the same time, so we pnrsul'd his simpll'r method of amniocentl'sis for this diaRnosis and prognostication. Alvarl'z, using phase microscopy to study hi~ biopsy material, pointed out that with p:-ogressi\'t' erythroblastosis there was a histolog:c ch ~ ng(' of the placenta to the immature state, that mark('d ~yncytial proliferation occurrrd, and that nl'W syncytial sprouts developrd. We studied sevl'ral of the placentas from our fetusl's that had hydrops as a rl'sult of Bart's hrmoglobin, both undrr phase microscopy and using vital staining, and found that thl'y apprarl'd exartly like the hiopsil's from sevt'rely affrctt'd infants wht're tht' erythroblastosis and / or hydrops was due to Rh incompatibility. In Bart's h('moglobinopathy the anemia is not related to blood destruction by anti-
March I , 1968
:\111 .
J. Ohlt. & Gynec.
hody but it is related to hlood destruction. AI. though the hlood d .. strut:tioll is g<'netically engl'nd.' red th., anemias are just as Sl'vere and the pathol"KY is the , .. me in both instances. I alii II lit as p"ssilllistit: as thl' previous two dis(,lIssants ;lIId I do hope that Dr. Hutchinson has not hl'l'n dis('I)\II'a~l'd to the point of discontinuing th(' study. I fl'rl that he has not been SlllTl'ssful IWl'aUSl' hl' has nut dl'\'e\opl'd a severe .'nollgh dl'gfl'l' of anemia in his experimental 'lIIimals to bring ahollt a tl'llr erythroblastic I"l'SpOnsl'. I furthl'!, fel'! that hydrops is probably a catadpllIil' tyPt' of t'n'nt which Occurs in hours or prohably not longt'f than a period DE days. Tht'fl' was onl' most int,'resting finding as far as I was conccrned, and do not know whether it impn'sS/'d him or not , and that was the fact that in all of his f"t,,1 lamhs the total serum protein was risinl!;. Could this Ql' a mechanism to replat't' t hI' hlood \'0111111<' lost? If he had persisted, how,'\'('r. in innl'asing thl' anemia in his fetuses, I helil'\'r that this prott'in would have rl'wrsl'd itself as proll;fl'ssi\'l' anemia brgan to aft"l't th., liver ,lI1d inted.'re with protein synthesis and as progn'ssiw anoxia affected the heart and thl' vascular Ill'd, osmotic prrssure changes in the cirnllating systl'm must occur which then result in the gen{'ralizt'd hydrops. We get an erythroblastosis-like picture in other conditions such as syphilis and diabetes, Perhaps here there are changes that occur within the placental vasculature 50 that there is a decrease in, and interference with, the transfer of oxygen, The placenta compensates by enlarging, the fetal erythropoetic syst('ms respond with more cells but as the oxyg('n tension continues to fall t~ leVl'ls that arl' {'wntually damaging to vital struc. IIlI'rs, su('h as the liwr, the hrart, and the vas. ('ular Iwd, dl'gl'nl'rati\'e changes occur that reo suit in hydrops. I pr('di('t that if Dr. Hutchinson's \iv('r or bone marrow studies show any erythropoetic response, ht' will he ahle to produce hydrops jf he will continue to do the hleedinR at a progressive rate rather than at a constant rate. This is undoubtedly the mechanism in thl" casl's of blood incompatibility. Certainly his hl'moglobin and hemato. crit Il'wls must 1)(' dl'opPt'd to nllt<'h lower levels OWl' mu('h longer P<'riod~ of time. DR. HCTCHlN!'ON (Closing) . It was not my {'x(J<'ctation at th .. outsrt of this study that the results would br so totally negative. It may very well be that the right question ha! not been asked, The finding! associated with hydrops may
Volum.IOO Numb... 5
nOI be set:oll(Jary to anl'mia but may, in facl, represent an imlllulle respollse to maternal antigen. Gross and Illicroscopit: observation of plat:ental cot yledons has not l)('en very rl'vl'alillg. It is difficult 10 make comparative statements, bllt there was no histologic evidence of villus edema or of proliferalion of trophoblast or stroma. All lambs which wt'fe not macerated were autopsied. No significant differences were found in tIll' weight or appearance of fetal organs. Precis/' quantitative morphologic studit's were not dOlle. It does set'm possible that hypoproteinemia may be a very important factor in the development of anasarca and Sl'rous effusions. We considered thl' possibility of trying to suppress protein synthesis in the ft,tus amI plat·t'nta but rl'jected it hecause of our concern that we might produce diffuse ct'lllllar changes with the chemicals which might make it difficult to dt·termine the specific calise of any ohsl'rvl'd changl'. I really do nOI know how long onl' could maintain a significant hypoprotl'ilwmic statl' in the fetus. Ndtht'r do I know what tht' threshold of anemia is below which hydrops fetalis will consistently develop. Mr. Roul'lll' told me recently that he had observed Ihe characteristic features
Experimental production of toxemia
695
of hydrops wilh a fetal hemoglobin of 80 pel' cc'n!. I believe that in general the more severe the anemia, the more profound the tissue hypoxia and the more likely that there will be free fluid or massive edema. It is my understanding that the monkey expl'riments of Adamsons were very acute studies. We thought that we might be successful by prolonging the fetal bleeding with this more chronic approach. The hydrops associated with abnormal hemoglobins such as Bart's is most interesting. Apparently tissue hypoxia occurs at relatively high hemoglobin levels because the abnormal hemoglobin does not release oxygen properly. A possible mechanism of action of phenylhydrazine is Ihe production of some change in hemoglobin which will decrl'ase its oxygen-carrying capacity. A variety of conditions have been associated wilh hydrops ft,talis. Syphilis and diabetes might slIggpst a matPrnal influence. Fetal-fetal transfllsions and fetal-maternal bleeds suggest that fptal anl'mia may cause the syndrome. The hydrops associalC'd with erythroblastosis and with abnormal hemoglobin levels may well suggest some primary basic immune mechanism.