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Review: hemolytic disease of the newborn

Definition and Pathophysiology Gestational age of the when the titer Hemolytic disease of the newborn (HDN) results reaches a critical level. from sensitization of fetal RBCs by maternal IgG Development phase of the on the fetal alloantibodyThe is an organ that has separate RBCs (Table 2). maternal and fetal circulatory systems. The main func- Efficiency of the fetal reticuloendothelial system in tion of the placenta is to exchange gases, electrolytes, clearing sensitized RBCs. and nutrients between the fetus and mother. However, As fetal RBCs are destroyed, , both conju- when there is transplacental passage of maternal IgG gated and unconjugated, accumulates. The accumu- alloantibody to the fetus, HDN may occur. lation of lipid soluble unconjugated (indirect) Sensitized fetal RBCs may undergo accelerated bilirubin in the newborn may be a greater problem destruction both before and after delivery. This pro- than the and hypoxia, because of its potential cess contributes to the increased accumulation of the for causing neurologic dysfunction. products of RBC hemolysis, especially bilirubin, and RBC destruction and accompanying problems may to fetal anemia. The extent of RBC hemolysis may Table 2. Common RBC : strength on cord cells and implication range from subclinical to severe. The severity of HDN in hemolytic disease of the newborn (HDN) depends on the extent of hemolysis and how well the fetus compensates for the anemia.1 Blood group system Development Implicated antigens at birth in HDN As RBC destruction occurs, the fetus compensates by increasing RBC production in the bone marrow, Rh well developed yes MNSs well developed yes * kidney, liver, and spleen. Nucleated RBCs and reticu- Kell well developed yes locytes may appear in the peripheral circulation in Duffy well developed yes Kidd well developed yes increased numbers. As the fetus continues to increase Diego well developed yes RBC production, hepatosplenomegaly with portal Dombrock well developed yes (mild) Gerbich well developed yes (mild) hypertension, ascites, and hepatocellular damage may Cartwright Yta poorly developed no occur. In addition, severe edema may result (hydrops Ytb well developed no fetalis) as cardiac function decompensates. Resultant ABO weaker than adult yes P weaker than adult no ** organ failure can cause death in utero.2 Lutheran weaker than adult yes The severity of HDN may depend on a number of vel very weak or absent ? Lewis very weak or absent no factors including- Knops/Mccoy very weak or absent no * Concentration of IgG antibody crossing the Chido/Rodgers very weak or absent no Cost-Stirling/York very weak or absent no placenta. * Anti-M and -N rarely cause HDN. Subclass of the maternal antibody (Table 1). **Anti-P+P1+Pk (Tja) has caused HDN.

Table 1. Properties of lgG subclasses

Properties IgG1 igG2 igG3 igG4

Percentage of total IgG 60-80 10-25 a9 3-4 Fixes complement sometimes occasionally usually no Crosses placenta yes—early may see yes—some yes—after lgG1 yes—some high levels in cord more destructive Macrophage binding yes no yes no

96 IMMUNOHEMATOLOGY, VOLUME 9, NUMBER 4, 1993 Hemolytic disease of the newborn

persist at least as long as the antibody remains in the , , rupture of an ectopic preg- newborn's circulation. The half-life of IgG is 21 days; nancy, or chorionic villus sampling (CVS).8 therefore, RBC sensitization caused by maternal IgG antibody may continue for several months after birth.3 Implicated and Their Frequency Only IgG antibodies are known to cause HDN. The Metabolism of Bilirubin in the Fetus and antigen against which the antibody is directed must be Newborn well developed on the fetal RBCs for RBC destruction In utero, conjugated (direct, water-soluble) biliru- to occur (Table 2). Until 1968 the most frequent cause bin crosses the placenta, enters the maternal circula- of HDN was anti-D in an Rh-negative woman but, even tion, and is excreted by the mother. Following birth, then, observations suggested that moderation of this conjugated bilirubin is excreted by the newborn's phenomenon might be possible. In 1958 Levine9 doc- hepatic and renal systems. In adults, unconjugated umented that Rh-negative women with ABO-incom- bilirubin binds to , is transported to the liver, patible Rh-positive infants formed anti-D less often and is enzymatically converted to conjugated biliru- than Rh-negative women with ABO-compatible Rh- bin, which is then excreted in the urine and bile. positive infants. As early as 1950, Race and Sanger10 However, the newborn's liver is inefficient in conju- had postulated this phenomenon, hypothesizing that gating bilirubin because of the low activity of the RBCs sensitized with anti-A and/or anti-B were enzyme glucuronyl transferase. The unconjugated removed before being presented to the reticuloen- bilirubin binds to albumin, but if there is an inade- dothelial system. This hypothesis was demonstrated quate level of albumin for the amount of bilirubin, experimentally in 1961.11 unbound bilirubin will deposit in the infant's tissues, including the brain, causing . This may In 1968 a study was undertaken to determine the result in permanent central nervous system damage. frequency of immunization in Rh-negative women The severity of kernicterus is influenced by the con- with Rh-positive .l2 Only Rh-negative women centration of albumin, the presence of competing with an ABO-compatible Rh-positive infant were stud- substances that also bind to albumin, acidosis, and the ied. The study showed that after the first degree of prematurity (stage of liver development). with an Rh-positive infant, 8 percent of Rh-negative women made anti-D. After a second pregnancy, an Origin of Maternal Alloantibody additional 8 percent of the women made anti-D.12A Clinically significant alloantibodies may be pro- separate study, reported in 1975, showed that the vol- duced when foreign RBC antigens enter the circula- ume of a TPH had an effect on alloimmunization.13 tion through pregnancy or transfusion. In pregnancy, With an ABO-compatible fetus (which occurs in 80% small numbers of fetal RBCs enter the maternal circu- of ), the risk of anti-D formation is 3 per- lation via the placenta, although the exact mechanism cent with less than 0.1 mL TPH, but the risk rises to 25 of the fetal-maternal RBC exchange is not known.4 percent with a 0.25-1.0 mL bleed, and to 65 percent Abruptio placentae, placenta previa, Cesarean section, when the TPH is greater than 5 mL. About 75 percent manual removal of the placenta, or preeclampsia of the women studied had a TPH of less than 0.1 mL. increase the risk of transplacental hemorrhage The overall risk of anti-D formation in an ABO-com- (TPH).5 patible pregnancy was 12-15 percent, and in an ABO- The Kleihauer-Betke staining technique6 uses the incompatible pregnancy the risk was 2-3 percent.13 difference in staining properties of hemoglobin A The fact that the presence of passive antibody pre- (maternal) and hemoglobin F (fetal) to determine the vents formation of that particular antibody when the presence of both fetal and maternal RBCs in the individual is exposed to the antigen was first shown in maternal circulation. This method has shown that 1900. The observation did not have clinical implica- most RBC exchange occurs at delivery as the placenta tions until Rh immune globulin (RhIG),a concentrat- separates from the uterus, although TPH can occur ed form of anti-D, was licensed for Rh prophylaxis in during the entire pregnancy. In one study, 45 percent 1968. Vials of RhIG contain 300 µg or 150 µg of puri- of pregnant women had fetal cells in their circulation fied anti-D manufactured from human plasma. RhIG during the third trimester.' TPH also occurs after is given to Rh-negative women within 72 hours of

IMMUNOHEMATOLOGY, VOLUME 9, NUMBER 4, 1993 97 J. MARTIN

delivery of Rh-positive infants. For RhIG to be effective, affected and whether in-utero intervention may be it must be given before sensitization to the alloantigen needed. These steps include past pregnancy history, has occurred and in a dose sufficient to cover the quan- maternal antibody titer, ultrasound, CVS, monocyte tity of antigens to which the woman has been monolayer assay (MMA), antibody-dependent cell- exposed.14-19 RhIG should also be given after every mediated cytotoxicity (ADCC) testing, amniocentesis, instance in which a TPH might occur, including abor- PUBS, and/or fetal cardiac monitoring.1 tion, amniocentesis, and percutaneous umbilical Past pregnancy history is not a good predictor of blood sampling (PUBS). It should also be given after HDN, although the severity usually increases with suc- CVS if the pregnancy will be carried to term8 cessive pregnancies.3 Maternal antibody titer may Between 1968 and 1987, the use of RhIG caused Rh carry more weight in the first affected pregnancy. immunization to decrease considerably, but did not Ultrasound shows the placental and fetal liver size, the eradicate HDN caused by anti-D. This situation may be presence or absence of hydrops, and the fetal gesta- due, in part, to failure to inject RhIG postpartum or the tional age.1 CVS is a placental biopsy by which fetal tis- occurrence of primary immunization during pregnan- sue can be obtained for genetic studies beginning at cy Other reasons are failure to inject RhIG after abor- 8 weeks of pregnancy.8Antigen typing can be done on tion or amniocentesis, or an undetected massive TPH. this fetal tissue using a modified Ashby, microfluores- To determine how to prevent anti-D formation dur- cent, or rosette technique. MMAs and ADCC tests can ing pregnancy, antenatal trials were conducted.20 be used to help determine the “clinical significance” Initially, RhIG was given at 34 weeks. Further trials of the maternal antibody so that the severity of HDN showed that giving RhIG at 28 weeks was more effec- can be predicted. tive. When given at 28 weeks, the rate of anti-D Amniocentesis will detect fetal RBC breakdown formation dropped to 0.1 percent. products in the amniotic fluid. It is the most accurate Alloantibodies other than anti-D have played a test for predicting HDN severity in the third trimester. more obvious role in HDN since the introduction of A linear tracing of the amniotic fluid is done and plot- RhIG. Anti-c, which is now half as frequent as anti-D,21 ted. Bilirubin will show an absorbance peak at 450 has been reported as causing mild to severe HDN.22 nm. The difference between the absorbance expected Up to 40 percent of infants affected by anti-c need (based on fetal age) and that observed is used to pre- .21Anti-K1 has been reported as dict severity (Liley Chart or the Freda classification).3 causing very severe HDN, including PUBS allows direct access to the and in utero death.23For a list of antibodies that cause and is used at 17-40 weeks gestation. The fetal RBC HDN, see Figure 1. sample obtained can be used for determination of ABO, Rh, and other antigen types, direct antiglobulin Anti-D, -C, -c. -Cw,-Cx, -E, -e, -EW,-ce, -CeS,-Rh:32, -Goa, -Bea, test (DAT), hemoglobin, and genetic studies.’ -Evans, -LW Anti-K1, -K2, -K5, -K3, -K4. -K6. -K7 Management of the Alloimmunized Female Anti-Fya, -Fyb, -Fy3, -Jka, -Jkb, -Jk3 Anti-M, -N,-S, -s, -U, -Vw, -Far. -Mv, -Mit, -Mta, -Mur, -Hil, -Hut, -Ena Different types of therapy have been tried to sup- Anti-Wra, -PP1,Pk, -Lua,-Lub, -Lu9, -Dia, -Dib, -Coa press the titer of preexisting maternal alloantibody The Anti-Bi, -By, -Fra, -Good. -Rd, -Rea, -Zd use of stroma from Rh-positive RBCs,24,25 Rh Anti-Ata. -Jra, -Lan, -Ge promethazine hydrochloride,27 or RhIG28,29 have not Fig. 1. Antibodies reported as causing HDN. been very effective. Plasmapheresis30,31 or IVIG32,33 have been of possible benefit in selected cases. Early prenatal testing should result in the recogni- tion of the need for serologic monitoring and treat- Management of the Fetus ment of HDN. Perinatal serologic testing is essential A primary problem in HDN is management of the for early detection and intervention. severely affected fetus, who may become hydropic. In Manitoba, in the early 1940s, 50 percent of affected Determination of HDN Severity infants were only mildly affected and survived without If a woman has an alloantibody, several steps may treatment. Twenty-five percent were born in relative- be taken to predict how severely an infant will be ly good condition, but developed severe kernicterus

98 IMMUNOHEMATOLOGY, VOLUME 9, NUMBER 4, 1993 Hemolytic disease of the newborn

after birth. Of these, 90 percent died and 10 percent nization.38 To prevent augmentation, it is important suffered severe neurological damage. The remaining to give RhIG according to the recommended antena- 25 percent developed hydrops and died in utero, half tal and postpartum schedule. between 18 and 34 weeks gestation, and the remain- Studies have shown that, although RhIG is IgG anti- ing half after 34 weeks.1 Premature delivery and D and therefore crosses the placenta when given ante- (IUT) have reduced these natally, the amount of antibody entering the fetal risks. IUT can be done with an intraperitoneal (IPT) circulation compared to the number of Rh-positive or intravascular technique. IVT is more difficult antigen sites is too small to cause a problem in the technically, but can be used for the moribund fetus.1 fetus. Some Rh-positive infants are born with a weak- ly positive DAT, and anti-D can be eluted from the Management of the Affected Newborn RBCs even though there is no evidence of RBC The risk of kernicterus has been reduced by the use destruction.' of exchange transfusion. In this procedure, bilirubin, In the United States, RhIG is prepared by the Cohn maternal antibody, and sensitized infant RBCs are cold ethanol method. RhIG can only be given intra- removed and replaced by donor blood. The gestation- muscularly. It contains small amounts of IgA and IgM al age of the infant and the severity of HDN determine as well as trace amounts of other plasma proteins.1 the bilirubin level at which the exchange transfusion is There is one report of a severe anaphylactic reaction performed. Light promotes metabolism of bilirubin in after injection of RhIG to an IgA-deficient individual the skin to water-soluble compounds that are excret- who had anti-IgA.39 ed by the kidney. Phototherapy may be helpful in low- Purified anti-D can also be made from human plas- ering bilirubin levels in mildly affected infants.34 It can ma using an ion exchange column. This product does reduce the bilirubin level of an infant 1.5 to 2 mg/dL not contain any IgA or IgM and has a low concentra- every 12-24 hours. Phototherapy is not recommend- tion of total protein. The product is licensed in ed as the primary treatment, but as an adjunct35 in Canada and can be given intravenously.40 Currently, moderately and severely affected infants. several groups are looking at the production of RhIG using a monoclonal anti-D.41,42 Postpartum Testing of the Infant Great strides have been made in the treatment and It is not necessary to test a cord blood specimen diagnosis of HDN in a relatively short time period. This (such as performing a DAT) from all infants, although article contains a brief overview of the topic. Additional one should be collected and available. If a mother has information can be found in Perinatal Transfusion an alloantibody or her physician suspects HDN based Medicine, published by the American Association of on clinical observations at delivery, testing should be Blood Banks in 1990.43 performed on cord blood.36 If the infant has had an IUT, test results must be interpreted with that in mind. References 1. Bowman J. Historical overview: hemolytic disease of the Prevention of Rh Alloimmunization newborn. In: Kennedy MS, Wilson SM, Kelton JG, eds. The protective effect of RhIG is dose-dependent. It Perinatal transfusion medicine. Arlington, VA: American Association of Blood Banks, 1990:1-52. is important to give the correct dose ifa massive fetal- 2. James LS. Shock in the newborn in relation to hydrops. Ann maternal hemorrhage (FMH) occurs. Studies show Obstet Gynecol1971;92:599-601. that 300 µg of RhIG will give protection for up to a 30 4.3. Walker RH, ed. Technical manual. 10th ed. Arlington, VA: American Association of Blood Banks, 1990:377-410. mL whole blood fetal bleed. The frequency of an FMH Cohen F, Zuelzer WW. Mechanisms of isoimmunization. II. of more than 30 mL has been calculated to be approx- Transplacental passage and postnatal survival of fetal imately 0.3 percent.' Rh immunization can be pre- erythrocytes in heterospecific pregnancies. Blood 1967; vented in this group by the routine use of a 30:796-804. 5. Lloyd LK, Miya F, Hebertson RM, et al. Intraparrum fetal postpartum screening procedure for fetal RBCs in the in Rh negative women. Obstet Gynecol 1990; maternal circulation. If positive, a quantitative test is 56:285-5. performed and the calculated dose of RhIG is given for 6. Kleihauer E, Braun H, Betke K. Demonstration von fetalem hämoglobin in den erythrozyten eines blutauastrichs. Klin prophylaxis.37 It has been suggested that an insuffi- Wochenschr 1957;35:637-8. cient dose of RhIG actually augments primary immu- 7. Bowman JM, PollackJM, Penston LE. Fetomaternaltranspla-

IMMUNOHEMATOLOGY, VOLUME 9, NUMBER 4, 1993 99 cental hemorrhage during pregnancy and after delivery. 31. Robinson EAE,Tovey LAD. Intensive plasma exchange in the Vox Sang 1986;51:117-21. management of severe Rh disease. Br J Haematol 1980; 8. Warren RC, Butler J. Morsman JM, et al. Does chorionic 45:621-3 1. villus sampling cause FMH? Lancet 1985;1:691. 32. Berlin G, Selbing A, Ryden G. Rhesus haemolytic disease 9. Levine P. The influence of the ABO system on Rh hemolytic treated with high-dose intravenous immunoglobulin Getter). disease. Hum Biol 1958;30:14-28. Lancet 1985;1:1153. 10. Race RR,Sanger R. Blood groups in man. Oxford: Scientific 33. de la Camara C, Arrieta R, Gonzalez A, et al. High-dose intra- Publications, 1950. venous immunoglobulin as the sole prenatal treatment for 11. Stern K, Goodman HS, Berger M. Experimental isoimmu- severe Rh immunization. N EnglJ Med 1988;318:519-20. nization to hemoantigens in man. J Immunol 1961; 34. Tabb PA, Inglis J, Savage DCL, Walker CHM. Controlled trial 87:189-98. of phototherapy of limited duration in the treatment of phys- 12. Woodrow JC, Donohue WTA. Rh immunization by pregnan- iological hyperbilirubinemia in low-birth-weight infants. cy: results of a survey and their relevance to prophylactic Lancet 1972;2:1211. therapy. Br MedJ 1968;4:139-44. 35. Hill RM, Meza GC.Utilization of blood components in neona- 13. Bowman JM. Rh erythroblastosis fetalis. Semin Hematol tal medicine. In Bell CA,ed. A seminar on perinatal blood 1975;12:189-207. banking. Washington, DC: American Association of Blood 14. Ascari WQ, Levine P, Pollack W. Incidence of maternal Rh Banks, 1978:109-30. immunization by ABO compatible and incompatible preg- 36. Judd WJ, Luban NLC, Ness PM, et al. Prenatal and perinatal nancies. Br MedJ 1969;1:399-401. immunohematology: recommendations for serologic man- 15. Freda VJ, Gorman JG, Pollack W. Successful prevention of agement of the fetus, newborn infant, and obstetric patient. experimental Rh sensitization in man with an anti-Rh gamma Transfusion 1990;30:175-83. 2-globulin antibody preparation: a preliminary report. 37. Widmann FK, ed. Standards for blood hanks and transfusion Transfusion 1964;4:26-32. services. 14th ed. Arlington, VA: American Association of 16. Zipursky A, Israels LG. The pathogenesis and prevention of Blood Banks, 1991. Rh immunization. Can Med Assoc J 1967;97:1245-57. 38. Pollack W, Ascari WQ, Kockesky RJ, et al. Studies on Rh pro- 17. Combined Study. Prevention of Rh-haemolytic disease. Br phylaxis I. Relationship between doses of anti-Rh and size of Med J 1966;2:901-14. antigenic stimulus. Transfusion 1971;11:333-9. 18. Clarke CA, Donohoe WTA, McConnell RB, et al. Further 39. Rivat L, Parent M, Rivat C. Accident survenu aprés injection experimental studies in the prevention of Rh-haemolytic de gamma-globulines anti-Rh du à la présence d’anticorps disease. Br Med J 1963;1:979-84. anti-IgA (letter). Press Med 1970;78:2072. 19. Chown B, Duff AM, James J, et al. Prevention of primary Rh 40. Bowman JM, Friesen AD, Pollock JM, et al. WinRho: Rh immunization: first report of the Western Canadian trial, immune globulin prepared by ion exchange for intravenous 1966-1968. Can Med Assoc J 1969;100:1021-4. use. Can Med Assoc J 1980;123:1121-5. 20. Bowman JM. Chown B, Lewis M. et al. Rh-isoimmunization 41. Crawford DH, Barlow MJ, Harrison JF, et al. Production of during pregnancy: antenatal prophylaxis. Can Med Assoc J human monoclonal antibody to rhesus D antigen. Lancet 1978:118:623-7. 1983;1:386-8. 21. Wenk RE, Goldstein P, Felix JK. Alloimmunization by hr'(c), 42. Bron D, Feinberg MB, Teng NNH, et al. Production of human hemolytic disease of newborns, and perinatal management. monoclonal IgG antibodies against rhesus (D) antigen. Proc Obstet Gynecol 1986;67:623-6. Natl Acad Sci (USA) 1984;81:3214-7. 22. Powell PJ, Brown SE, Dike AE, Inskip MJ. The significance of 43. Kennedy MS, Wilson SM, KeltonJG, eds. Perinatal transfusion anti-c alloimmunization in pregnancy. Br J Obstet Gynecol medicine. Arlington, VA: American Association of Blood 1986;93:1044-8. Banks, 1990. 23. Farr V, Gray E. Pregnancy outcome in mothers who develop Kell antibodies. Scot MedJ 1988;33:300-3. 24. Biermé SJ, Blane M, Abbal M, et al. Oral Rh treatment for Judith Martin, MT(ASCP)SBB, Director Reference severely immunized mothers. Lancet 1979;1:604-5. Laboratory,American Red Cross Blood Services, 25. Barnes RMR, Duguid JKM, Roberts FM, et al. Oral adminis- Badger Region, P.O. Box 5905, Madison, WI53705- tration of erythrocyte membrane antigen does not suppress anti-Rh(D)antibody responses in humans. Clin Exp Immunol 0905 1987;67:220-6. 26. Carter BB. Preliminary report on a substance which inhibits anti-Rh serum. AmJ Clin Pathol1947;17:646-9. 27. Gusdon JP, Caudle MR, Herbst GA, Iannuzzi NP. Phagocytosis and erythroblastosis: I. Modification of the neonatal response by promethazine hydrochloride. Am J Obstet Gynecol 1976:125:224-6. 28. Bowman JM. PollockJM. Reversal of Rh alloimmunization. Fact or fancy? Vox Sang 1984:.17:209-15. Notice to Readers: All articles published, 29. De Silva M,Contreras M, Mollison PL. Failure 0f passively) including communications and book reviews, administered anti-Rh to prevent secondary Rh immune reflect the opinions of the authors and do not responses. Vox Sang 1985;48:178-80. 30 Graham-Pole J, Barr W, Willoughby MLN. Continuous flow necessarily reflect the official policy of the plasmapheresis in management of severe Rhesus disease. Br American Red Cross. MedJ 1977;1:1185-8.

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