ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 12, No. 5 Copyright © 1982, Institute for Clinical Science, Inc.

Laboratory Identification of Erythroblastosis FetalisELAINE M. KURTZ, M.D., ALEXANDROS A. PAPPAS, M.D.,

and ALBERT CANNON, M.D.D epartm ent o f Laboratory Medicine,

Medical University o f South Carolina, Charleston, SC 29403

ABSTRACTHemolytic disease of the newborn (HDN) is a disease of the fetus and

newborn owing to fetal-maternal blood group incompatibility. It may present as a barely perceptible hemolytic process or manifest the severe symptoms of hydrops fetalis. The clinical laboratory assumes the role of supplying data needed for the diagnosis, treatm ent and prevention of the disease. Prenatal immunohematologic testing identifies those women whose fetus is at risk for Rh-HDN and may indicate those patients who should have amniocentesis. Analyses of amniotic fluid identifies those fetuses which need intrauterine transfusion. Postnatal studies can confirm suspected HDN, identify those neonates who are at risk of developing kem icturus, and determ ine which neonates may need exchange transfusion or phototherapy. Maternal prenatal and postnatal testing identifies those females eligible for Rh-immune globulin therapy to prevent HDN.

IntroductionErythroblastosis fetalis or hemolytic

disease of the newborn (HDN) is a disease of the fetus and neonate which is charac­terized by a hemolytic anemia with vary­ing degrees of severity. The hemolytic anemia occurs as a result of maternal allo- antibodies which are transmitted to the fetus via the placenta. The important clin­ical manifestations are anemia, jaundice, hepatosplenomegaly and irreversible damage to neurons of the central nervous system, especially those of the basal gang­

lia (kernicterus). The severity of the dis­ease varies widely from a slight anemia and hyperbilirubinem ia at birth to intra­uterine death in the third trimester.46Rh-Hemolytic Disease of the Newborn (Rh-HDN)

The syndrome of erythroblastosis had been recognized for many centuries.2 However, it was not until the discovery of the Rh blood group system in 1939 and 1940 with the detection of the antibody against the antigen D (also known as Rh0)

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LABORATORY ID EN TIFIC A TION O F ERYTHROBLASTOSIS FETALIS 389that the basic pathogenesis of erythro­blastosis fetalis was established.28,31 Clas­sically hemolytic disease of the newborn (HDN) owing to anti-D occurs in D nega­tive women who have been previously sensitized to the D antigen. In the vast majority of cases, this sensitization to the D antigen with the production of anti-D antibody is the result of a previous preg­nancy in which the fetal red cells con­tained the D antigen. The sensitizing dose of fetal red cells most commonly oc­curs at the time of delivery as a result of a fetal-maternal bleed. The ABO type of the mother and fetus is an ameliorating factor with regards to the developm ent of mater­nal sensitization.29,30 Type ABO incom­patible fetal red cells are often destroyed before maternal sensitization to the D antigen has time to take place. On rare occasions, maternal sensitization to the D antigen can occur during the first preg­nancy and can result in HDN.39 Maternal sensitization to the D antigen can also re­sult from a transfusion of D positive blood, spontaneous or induced abortions, and ectopic pregnancies.

IgG is the only immune globulin which is transported across the normal pla­centa.8,17,20 At 33 weeks of gestation, the maternal and fetal levels of IgG are ap­proximately equal,18 and at term the fetal level may exceed the maternal level.17 Antibodies directed against antigens of the Rh system are of the IgG type. The subclasses IgG l and IgG3 bind more effi­ciently than IgG2 and IgG4 to splenic macrophages, thereby readily inducing the destruction of immune globulin coated erythrocytes. Infants with red cells coated with IgG3 statistically have a much more severe form of hemolytic disease of the newborn while infants with IgG l have a HDN of variable severity.11 Meth­ods are available for differentiating the IgG subclasses.12,21,35,51

The frequency of pregnancies in the general population which theoretically can result in Rh-HDN (Rh negative moth­

ers and Rh positive fetus) is 10 percent in Caucasians, five percent in Negroes, and one percent in Orientals.4 Prior to the ad­vent of Rh immune globulin, only about 12 percent of Rh negative mothers be­came sensitized.1,15 Type ABO incompati­bility, poor antibody formation by the mother or an insufficient antigenic stimu­lus (small volume of fetal-maternal bleed) protect Rh negative mothers from sensiti­zation.6,41,45 With the extensive use of Rh immune globulin to prevent maternal sensitization to the D antigen, the inci­dence of Rh sensitizations has fallen to one percent.15

The most common cause of HDN is ABO incompatibility. However, the most common cause of severe fetal hemolytic anemia requiring exchange and possibly intrauterine transfusion is maternal anti- D. Maternal antibodies to Kell, C, and E can cause severe hemolytic disease, but are relatively rare.55 It is essential to estab­lish the potential for fetal hemolytic disease as early as possible so that the pregnancy can be properly managed and appropriate therapy undertaken.

The diagnosis of HDN begins with a complete history and physical examina­tion at the first obstetric visit. Particular attention must be paid to the course and outcome of previous pregnancies (abor­tions, stillbirths) and to any prior trans­fusions. Immunohematologic testing should include ABO grouping of mother and fa­ther, maternal Rh typing, and an antibody screen.

The results of the ABO grouping will indicate if the fetus is at risk for develop­ing ABO-HDN. Rh typing will identify the Rh negative women, who are at risk for becoming sensitized to the D-antigen. Antibody screens should be run on both Rh positive and Rh negative women to detect other antibodies which may cause HDN or create compatibility problems if a fetal or maternal transfusion is needed at delivery or post-partum. Rh negative women with negative antibody screens at

3 9 0 KURTZ, PAPPAS, AND CANNON

the initial visit should have repeat studies at 28, 32, and 36 weeks gestation to detect an early anamnestic (secondary immune) response.52 If these repeat studies are neg­ative, no further testing during that preg­nancy is indicated.

If the prenatal antibody screen is posi­tive, the antibody must be identified. The antibody should be classified as to w hether it is of IgG or IgM type (IgM does not cross the placenta) in order to deter­mine if the antibody is clinically signifi­cant.23,35 If the antibody is clinically sig­nificant, the serum sample from the initial visit should be properly frozen and later titered in parallel with subsequent serum samples from the patient.

The antibody titer procedure consists of serial dilutions of maternal serum which are reacted with a suspension of group O RBC’s that possess the suspected antigen (usually D). The titer with the highest di­lution at which hemagglutination occurs is reported. A two-tube antibody rise is regarded as a significant increase.

In a pregnancy in which a clinically sig­nificant antibody (usually anti-D) is dem­onstrated, rising serial antibody titers usu­ally indicate the fetal erythrocytes possess the corresponding antigen and, therefore, the fetus is likely to suffer from hemolytic disease.48 The antibody titer, in general, does not correlate well with the severity of HDN.40 The primary value of an anti­body titer is to identify patients who should have amniocentesis.

Amniocentesis provides the clinician with a means to evaluate the degree of intrauterine fetal hemolysis taking place. It is also useful to assess the overall condi­tion of the fetus. Amniocentesis is indi­cated when the clinically significant ma­ternal antibody titer is 1:32 or greater in the fourth or fifth month of preg­nancy.38'48,55 If there is a history of a pre­vious pregnancy complicated by HDN, amniocentesis is indicated regardless of the present maternal serum antibody

titer.38 The critical antibody titer value that is associated with the need for amnio­centesis must be determ ined by each lab­oratory. The initial amniocentesis is gen­erally performed betw een the 28th and 32nd week of pregnancy.48 Amniocentesis may be repeated as often as necessary up to the time of delivery. Complications of amniocentesis include an increase in ma­ternal Rh sensitization from fetal-mater­nal bleeding, infection, and premature la­bor.3,48 However, these complications are relatively infrequent.

Amniotic fluid is normally straw col­ored and slightly turbid. Inspection of amniotic fluid upon receipt in the labora­tory can be quite helpful. Yellow, slightly turbid amniotic fluid may indicate an Rh positive fetus affected with HDN; red, opaque fluid indicates blood contamina­tion; green, opaque fluid indicates meconium staining; and yellow-brown opaque fluid is often associated with in­trauterine death of the fetus.47

Bilirubin, a metabolic by-product of physiologic hemolysis, is present in very low concentrations in the amniotic fluid during the first trim ester of pregnancy. Normally, as erythropoiesis accelerates, bilirubin levels in the amniotic fluid in­crease until the 20th to 26th weeks. In­creased abnormal levels of amniotic fluid bilirubin give an indication as to the se­verity of hemolysis in the fetus.5,54

Normal amniotic fluid shows a specific spectral absorbance pattern when scanned from 350 nm to 750 nm. A spectral scan of amniotic fluid containing bilirubin will show a characteristic change in absorb­ance or optical density (O.D.) character­ized by a rise at 375 nm, a peak at 450 nm, and a return to the baseline at 525 nm (figure 1). Transposing the spectral scan to a semilogorithmic scale (abscissa, wave­length in linear scale; ordinate, AO.D. in log scale) results in linearization of the scan results. The net difference between the peak absorbance at 450 nm and the

LABORATORY ID EN TIFIC A TIO N O F ERYTHROBLASTOSIS FETALIS 3 9 1

theoretic baseline taken betw een 375 nm and 525 nm is the absorbance contributed by bilirubin or the AO.D. 450.13,33

Since sunlight and artificial lighting de­stroy bilirubinoid pigment rapidly, amni- 3otic fluid must be placed immediately in -amber or foil wrapped test tubes. In the gspectral scan of amniotic fluid, potential gtechnical problems can arise from con- £tamination owing to blood or meconium (figure 2). Hemolyzed blood gives the characteristic peaks of oxyhemoglobin at 415 nm, 540 nm, and 757 nm. Hemolysis tends to decrease the net absorbance at 450 nm; hence, the severity of hemolytic disease is underestimated. Meconium staining of amniotic fluid is indicative of fetal distress. Meconium typically peaks at about 405 nm and slowly recedes to baseline at about 550 nm.

Predictive interpretations of fetal prog­nosis can be made based on the relative concentration of bilirubin at various stages of gestation.10,14,34 Liley, plotting the AO.D. at 450 nm versus gestational age, defined three major prognostic zones32 (figure 3). Zone III includes intra­uterine deaths, neonatal deaths, and in­fants with low cord hemoglobin levels. Fetuses with values in Zone III, before the 32nd week, require intrauterine trans­fusion to prolong intrauterine survival un­til viability. After the 32nd week, Zone III values indicate the need for immediate delivery to prevent fetal death. Zone II values indicate the need for repeat analy­sis and possible early delivery. Zone I values indicate a good prognosis with re­gards to HDN in a near term fetus.

Serial amniotic fluid analyses will pro­vide the clinician with an indication of any possible adverse trends. An increas­ing or stationery AO.D., depending on the gestational age and clinical circum­stances, will indicate the type of interven­tion needed. This may be exchange trans­fusion or simply following the fetus until delivery.

W AVELENGTH (nm)

F ig u r e 1. Calculation of AO.D. at 450 nm. The spectral scan of amniotic fluid containing bilirubin will show a characteristic change in absorbance at 450 nm. The net difference between the peak ab­sorbance at 450 nm and the theoretical baseline drawn between 375 nm and 525 nm is the absorb­ance contributed by bilirubin or the AO.D. 450.

It has been recommended that erythro­cytes from amniotic fluid be ABO grouped, Rh typed ,44 and that an acid elution test be performed to determ ine if

F ig u r e 2. Spectral scan of amniotic fluid. Nor­mal amniotic fluid is represented by A; amniotic fluid containing bilirubin is represented by B; amni­otic fluid containing oxyhemoglobin is represented by C; and amniotic fluid containing meconium is represented by D.

3 9 2 KURTZ, PAPPAS, AND CANNON1. 0 -

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£c0IO1 0.10- q : o'<

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F i g u r e 3. Nomogram of AO.D. 450 nm against gestational age. Liley correlated the AO.D. 450 nm with the severity of hemolytic disease. (Liley, A. W.: Errors in the assessment of hemolytic disease from amniotic fluid. Amer. J. Obstet. Gynecol. 86:485- 494, 1963.)

the cells are of fetal or maternal origin.24 This procedure is neither reproducible nor a standard technique and does not aid in the management of the patient.

Amniotic fluid creatinine (to assess fetal weight) and phospholipid determinations (to assess fetal lung maturity) will aid the clinician in the determination of the po­tential viability of the fetus that needs im­mediate delivery. Maternal estriol, hu­man chorionic gonadotropin (HCG), and human placental lactogen (HPL) will aid in the assessment of placental function. These tests provide an indication of fetal maturity or fetal distress in a pregnancy complicated by Rh-HDN.55

Intrauterine transfusions may be the only hope for a fetus with severe hemo­lytic disease. The timing of the procedure is crucial. With respect to gestational age, 91 percent of fetuses died in utero when the initial intrauterine transfusion (IUT) was given before the 25th week of gesta­tion, 76 percent died when IUT was given betw een the 25th and 27th week, and 44

percent died when IUT was given at the 28th week or later.48,53 After the 32nd week, the chance of fetal survival follow­ing delivery is good and intrauterine transfusion is not generally indicated. Possible fetal complications of IUT in­clude tissue trauma, hepatitis, and bacte­rial infection. Possible maternal compli­cations include premature rupture of the membrane, infection, and bleeding.48,53

At birth, hemolytic disease can present as a barely perceptible hemolytic process identified only by immunohematologic testing. The most severe symptoms form a syndrome referred to as hydrops fetalis. This includes severe anemia with heart failure, hepatosplenomegaly, and marked generalized edema with epidermal ex­foliation. The postpartum laboratory diag­nosis of HDN is made on cord blood and should include:

(1) Direct antiglobulin test (D.A.T.), (2) Hemoglobin determination, (3) Differen­tial cell count on a blood smear stained with Wright’s stain, (4) Absolute reticulo­cyte count, (5) Indirect serum bilirubin,(6) Reserve albumin binding capacity, and (7) ABO group and Rh type.

The presence of maternal anti-D anti­bodies in an infant with a positive direct antiglobulin test (Coombs), low umbilical cord hemoglobin concentration, and an elevated indirect serum bilirubin is virtu­ally diagnostic of HDN. In severely af­fected infants, the antiglobulin test almost always gives a strongly positive result. However, the strength of the direct anti­globulin test is not directly correlated with the severity of the disease.39

Cord blood hemoglobin concentration is an extremely valuable param eter in as­sessing the severity of the hemolytic ane­mia.39 Greater than 50 percent of affected infants have a cord hemoglobin level above 14 mg per dl, which is usually ac­cepted as the lower limit of the reference range. Normally the hemoglobin level of the newborn falls within the first 72 hours of life, but in HDN the decrease in hemo­

L A B O R A TO R Y ID E N T IF IC A T IO N O F E R Y T H R O B L A S T O S IS F E T A L IS 393

globin concentration is precipitous and the signs and symptoms of anem ia b e ­come apparent. Infants with the clinical syndrome of h y d r o p s f e t a l i s at birth may have um bilical cord hem oglobin levels as low as 3 g per dl. Such unusually low hem oglobin concentrations may be due to technical errors as a result of dilution of the cord blood by the marked tissue edem a fluid. These hydropic infants, if they do not die in u te ro , usually do not survive the neonatal period.

The most characteristic findings on a W right’s stained thin smear of the cord blood is the trem endous num ber of nucle­ated macrocytic erythrocytes (figure 4). These macrocytic red cells appear in all stages of maturation, hence the original name of the disease “erythroblastosis fetalis.” The erythrocytes may be normo­chromic or polychromatophilic. The retic­ulocyte count is often markedly elevated reflecting the bone marrow’s response to anem ic stress. There may be a marked leukocytosis in the severely affected neo­nates. Throm bocytopenia with accompa­nying purpura may likewise occur in se­verely affected neonates. D issem inated intravascular coagulation may occur com­

plicating the morphologic picture of the peripheral blood smear.56

The anem ia should be treated by ex­change transfusion. This will rem ove the antibody-coated erythrocytes from the neonate, incom patible antibody, and the intravascular indirect bilirubin. With im­provem ent of the oxygen carrying capac­ity of the blood, symptoms of congestive heart failure are thereby relieved. There is clinical and pathologic evidence that the degree of neuronal damage of the basal ganglia of the brain by-products of b ilirubin metabolisms is related to the se­verity of the anemia. An increase in the perm eability of the blood brain barrier owing to hypoxia with resulting cerebral edem a creates an environm ent for severe and perm anent CNS damage.

A significant problem of hemolytic dis­ease of the new born is hyperbilirubin­em ia and potential bilirubin encephalo­pathy (kernicterus). Bilirubin in the fetus is normally transferred across the placenta to the m aternal circulation, is conjugated, and excreted by the maternal liver. In nor­mal neonates, serum bilirubin rises dur­ing the first few days of life, secondary to physiologic red cell destruction and also

F i g u r e 4. Im m a tu re re d b lo o d c e lls in p e r ip h e ra l b lo o d in s e v e re e ry th ro ­b la s to s is . N ote: ru b rib la s t, p ro ru b r ic y te , ru b r ic y te , a n d m e ta ru b r ic y te s .

394 KURTZ, PAPPAS, AND CANNON

to a deficiency of hepatic glucuronyl transferase. This enzyme conjugates bilirubin to a water-soluble excretable form. In infants with HDN, bilirubin me­tabolism is further compromised by ab­normally increased red cell destruction. The indirect bilirubin level that will produce kernicterus is not clearly de­fined.19 In full term infants with HDN, bilirubin above 20 mg per dl has been accepted as the level for significant risk of kernicterus.

Indirect bilirubin is bound to albu­min. The fraction of indirect bilirubin not bound or loosely bound by albumin may be called the “free bilirubin” frac­tion. This “free bilirubin” fraction crosses the blood brain barrier and causes neur­onal toxicity.42 The determination of the reserve albumin binding capacity (R.A.B.C.) has been used to develop indi­ces at which kernicterus may develop and exchange transfusion is indicated.7,26,48

No universal criteria have been estab­lished regarding when exchange transfu­sion should be done.43,48,55 However, an immediate exchange transfusion is usu­ally indicated in a mature neonate with a cord hemoglobin less than 12.0 mg per ml and an indirect bilirubin concentration greater than 4.5 mg per dl.36 With compli­cations such as prematurity, hypoxia, in­fection, and acidosis, the risk of develop­ing kernicterus at lower levels of bilirubin concentration is significant.16

For neonates not fulfilling the criteria for exchange transfusion, continuous phototherapy is utilized.22 Serum bili­rubin levels are measured every eight to ten hours until the serum bilirubin level starts falling. This generally occurs be­tween the third and sixth post-partum day.ABO—Hemolytic Disease of the Newborn (ABO-HDN)

Type ABO incompatibility is the most common cause of hemolytic disease in the fetus and neonate.7 Most cases are not of clinical importance and do not require

treatment. The hemolytic process due to ABO incompatibility often occurs in the first born, in contrast to the hemolytic pro­cess produced by fetal-maternal Rh in­compatibility. Type ABO-HDN almost al­ways involved mothers of group O, and neonates of groups A, B, or AB.25,49,50 The pathophysiology is essentially the same as that of HDN produced by antibodies to erythrocytic antigens of the Rh system.

The diagnosis of ABO-HDN is almost always made after delivery and is often only made after excluding all other possi­ble causes of early post-partum hyperbili­rubinemia. Type ABO-HDN cannot be predicted by any antenatal testing since the demonstration of immune anti-A or anti-B in the maternal serum has little pre­dictive value as to the presence or severity of the hemolytic process.

The ABO group and Rh type should be determ ined on any neonate who develops hyperbilirubinem ia shortly after birth. A direct antiglobulin test performed on the neonate’s erythrocytes should also be done. If the direct antiglobulin test is positive, the mother’s serum should be checked against a red cell panel and the specificity of her antibody determined. If the reaction of the direct antiglobulin test is weakly positive, the mother and infant are usually ABO incompatible. However, antibodies other than anti-A and anti-B may also be present and, as stated pre­viously, should be identified and their clinical significance determined.

The most satisfactory test to detect fetal- maternal ABO incompatibility is an elu­tion of the ABO antibody from the affected infant’s red cells.27 The eluate is then tested against adult A and B red cells.57 The detection of free antibody in the se­rum of ABO incompatible infants can be demonstrated and may substantiate the diagnosis of ABO incompatibility.58

Hematologic findings in fetal-maternal ABO incompatibility usually include a mild degree of anemia50 associated with slight reticulocytosis and spherocytosis.

LABORATORY ID EN TIFIC A TION O F ERYTHROBLASTOSIS FETALIS 3 9 5

The most common manifestation of ABO disease is jaundice. It appears dur­ing the first 24 hours of life. It rarely, if ever, is severe enough to be associated with the developm ent of kemicterus. Treatment, w hen necessary, consists of phototherapy. Rarely an exchange trans­fusion may be necessary. Serial indirect serum bilirubin concentrations and re­serve albumin binding capacity are used to determ ine the necessity and type of therapy. Fresh, saline washed, packed erythrocytes which are group O are used if an exchange transfusion is indicated.Other Blood Antigen Induced HDN

Although anti-D and the ABO system antibodies are the most frequent causa­tive antibodies of HDN, other antibodies in the Rh system, Duffy, Kidd, Kell and other systems can cause a hemolytic pro­cess of varying severity if the antibody can cross the placenta in sufficient concentra­tion and gain access to the fetal circula­tion. Severe HDN has been reported by antibodies directed against low incident antigens.39 Antibodies directed against high incident antigens also cause HDN.39 The same principles normally involved in prenatal testing and neonatal testing ap­ply with these antibodies to identify those cases at risk and to provide therapy as are used in Rh-HDN.Prevention of Rh-HDN

In women who are already alloimmu- nized, no practical means are available to suppress clinically the concentration of circulating maternal alloantibody. The methods used to control antibody forma­tion in organ transplant cases and auto­immune disease states are not applicable. Plasmapheresis can reduce a circulating maternal alloantibody on a short-term ba­sis only.

The usual candidates for Rh-immune globulin prophylaxis are women who are not alloimmunized to the D-antigen and

who are D and D “ negative and who have delivered a D or D u positive infant. If the direct antiglobulin test of the cord blood is positive, an elution test should be per­formed and the antibody identified. Rh- immune globulin should be given in such cases unless the antibody is identified as anti-D.4,38,39

An important dosage consideration in the prophylactic administration of Rh-im­mune globulin is the volume of the fetal- maternal bleed. The detection and quan­titation of the fetal-maternal hemorrhage can be determ ined by an acid-elution technique. Even though the technique is relatively simple, the test should be per­formed by experienced personnel in or­der to obtain reliable results.9,24 Rh-im- mune globulin is supplied as one ml of sterile, clear fluid. In the United States, a single dose of Rh-immune globulin, com­prising 300 ¡Jig IgG anti-D, is used to pre­vent immunization in cases where no more than 30 cc of fetal blood, or the equivalent of 15 cc of packed fetal cells, have entered the maternal circulation. The dose is generally adm inistered as an intramuscular injection within 72 hours after delivery. The failure rate in prevent­ing alloimmunization is approximately two percent.37

Ante partum administration of Rh- immune globulin has been advocated in Rh-negative women at risk who have amniocentesis, ectopic pregnancies, and spontaneous or induced abortions. The Rh-immune globulin reduces the inci­dence of fetal cells in the maternal circula­tion and causes no adverse reaction in the D positive fetus.4 In Rh-negative females who have received a transfusion with Rh positive blood, it is advisable to attem pt to prevent alloimmunization by the appro­priate use of Rh-immune globulin.Summary

The clinical laboratory assumes the paramount role of supplying accurate data

396 K U R T Z , P A P P A S , A N D C A N N O N

to the attending physician for the diagno­sis, treatm ent and prevention of HDN. Maternal prenatal testing identifies pa­tients at risk for Rh-HDN. The antibody titer is of primary value in assessing pa­tients as candidates for amniocentesis. Amniotic fluid analyses provide an assess­m ent of fetal prognosis in HDN and also an assessment of gestational age, lung ma­turity, and placental function. In severe HDN, amniotic fluid analysis can indicate the need for intrauterine transfusion. Postnatal laboratory studies can confirm the suspected diagnosis of HDN, identify those neonates at risk of developing kernicturus, and provide the physician with information pertaining to the treat­ment of HDN. Finally, prenatal and post­natal laboratory testing identifies those fe­males eligible for Rh-immune globulin therapy to prevent HDN in subsequent pregnancies.References

1. Al l e n , F . H .: Attempt at prevention of intra­uterine death in erythroblastosis fetalis. New Eng. J. Med. 269:1344-1349, 1963.

2. Al l e n , F. H., J r . and D i a m o n d , L. K.: Erythro­blastosis Fetalis. Little, Brown (Boston), 1958.

3. Al p e r n , M. W., C h a r l e s , A. G., and F r i e d ­m a n , E. A.: Amniocentesis in the management of Rh-sensitized pregnancies: Technique accu­racy of results and complications. Amer. J. Obstet. Gynecol. 95:1123-1128, 1966.

4. A s c a r i , W. Q.: Serology of erythroblastosis fetalis. Modern Management of the Rh Prob­lem, 2nd ed. Queenan, J. T., ed. Hagerstown, MD, Harper and Row, 1977.

5. Bevis, D. C. A.: Blood pigm ent in hemolytic disease of the newborn. J. Obstet. Gynecol. Brit. Comm. 63:68-75, 1956.

6. B o o t h , P . B., D u n s f o r d , J., G r a n t , J., and M u r r a y , S.: Hemolytic disease in first born infants. Brit. Med. J. 2:41-42, 1953.

7. B o w m a n , J. M.: Neonatal management. Mod­ern Management of the Rh Problem, 2nd ed. Queenan, J. T., ed. Hagerstown, MD, Harper and Row, 1977.

8. B r a m b e l l , F. W. R., H e m m in g s , W. A., O a k ­l e y , C. L., e T AL: The relative transmission of the fractions of papain hydrolyzed homologous gamma globulins from the uterine cavity to the fetal circulation in the rabbit. Proc. Roy. Soc. Biol. 151 ;478-482, 1960.

9. C o h e n , F ., Z u e l z e r , W. W., G u s t a f s o n , P.C ., and E v a n s , M. M.: Mechanisms of iso-im­

munization I. The transplacental passage of fe­tal erythrocytes in homospecific pregnancies. Blood 2 3 :6 2 1 -6 2 6 , 1964.

10. D i t o , W . R.: Amniotic fluid analysis in preg­nancy at risk. Chicago Amer. Soc. of Clin. Path.1975.

11. P i r o f s k y , B. and E n g e l f r i e t , C. P.: Hemo­lytic anemia. Progress in Immunology, vol. II. Brent, L. and Holborrow, J., eds. Amsterdam, North Holland, 1974 , pp. 2 9 7 -3 0 0 .

12. F r a n k o w s k a , K. and G o r s k a , B.: IgG Sub­classes at anti-Rh antibodies in pregnant women. Arch. Immunol. Ther. Exp. 2 6 :1 0 9 5 - 1098, 1978.

13. F r e d a , V. J.: The Rh problem in obstetrics and a new concept of its management using amnio­centesis and spectrophotometric scanning of amniotic fluid. Amer. J. Obstet. Gynecol. 9 2 :3 4 1 -3 7 4 , 1965.

14. F r e d a , V. J ., G o r m a n , J . G ., and P o l l a c k , W .: Rh factor: Prevention of isoimmunization and clinical trials on mothers. Science 151 :8 2 8 -8 3 0 , 1966.

15. F r e d a , V. J ., G o r m a n , J . G ., P o l l a c k , W ., and B o w e , E.: Prevention of Rh hemolytic dis­ease—Ten years clinical experience with Rh immuno globulin. New Eng. J. Med. 2 9 2 :1 0 1 4 - 1016, 1975.

16. G a r t n e r , L. M ., S n y d e r , R. N., C h a b o n , R. S., ET AL: Kernicterus: High incidence in prema­ture infants with low serum bilirubin concen­tration. Pediatrics 4 5 :9 0 6 -9 1 7 , 1970.

17. G i t l i n , D., Ku n a t e , J ., U r r u s t i , J., and M o ­r a l e s , C.: Selective and directional transfer of 75 gamma 2 globulin across the human pla­centa. Nature 2 0 3 :8 6 -8 7 , 1964.

18. G u d s o n , J. P .: Fetal and maternal immuno­globulin levels during pregnancy. Amer. J. Ob­stet. Gynecol. 103:8 9 5 -9 0 0 , 1969.

19. H a r p e r , R. G., S i a , C. G., and K i e r n e y , C. M.: Kernciturus 1980: Problems and practices viewed from the perspective of the practicing clinician. Clin. Perinatol. 7 :7 5 -9 2 , 1980.

20. H a r t l e y , P.: The effect of peptic digestion on the properties of diptheria toxin. Proc. Roy. Soc. Biol. 1 3 8 :4 9 9 -5 1 3 , 1951.

21 . H u n t , J. S ., B e c k , M . L., H a r d m a n n , J. T., e t AL: Characterization of human erythrocyte to alloantibodies by IgG subclass and monocyte interaction. Amer. J. Clin. Path. 7 4 :2 5 9 -2 6 4 ,1980.

22 . T a b b , P. A., Sa v a g e , D. C. L., I n g l is , J., and WALKER, C. H. M.: Controlled trial of photo­therapy of limited duration in the treatment of physiological hyperbilirubinem ia in low-birth- weight infants. Lancet 2 :1 2 1 1 -1 2 1 2 , 1972.

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