HEMOLYTIC DISEASE OF THE FETUS & NEWBORN

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1 HEMOLYTIC DISEASE OF THE FETUS & NEWBORN I. Definition - Red cell destruction in a fetus or newborn caused by feto-maternal blood group incompatibility. Maternal IgG antibody crosses the placenta and attaches to the corresponding antigen on fetal red cells II. Etiology A. Conditions for Hemolytic Disease of the Fetus and Newborn (HDFN) to occur: 1. The woman must be exposed, through pregnancy or transfusion, to a red cell antigen she lacks. 2. The antigenic exposure must cause immunization and production of antibody. 3. The antibody must be able to cross the placenta (IgG) and be of sufficient concentration to cause red cell destruction. a. Receptors on the Fc portion of the IgG molecule assist in its transfer across the placenta. b. IgG 1 and IgG 3 cause hemolysis more often than IgG 2 and IgG The infant must possess the antigen corresponding to the maternal antibody. B. Mechanism 1. Red cells from the fetus enter the circulation of the mother and stimulate production of antibody. 2. During subsequent pregnancy with an incompatible fetus, maternal antibody crosses the placenta and reacts with the antigen on the fetal red cells. 3. Sensitized cells are hemolyzed or destroyed by the RES in the fetus. C. Antigenic exposure during pregnancy 1. Small amounts of fetal cells may leak into maternal circulation during pregnancy. As little as 1 ml may be enough to immunize the mother. 2. Most common exposure to red cells in a woman is at the time of placental separation during delivery. CLS 422 Clinical Immunohematology I Page 1 of 10

2 3. The greater the volume of feto-maternal bleeding, the greater the chance of antibody production. 4. Feto-maternal bleeding can occur during pregnancy during amniocentesis, abortion, ectopic pregnancy, trauma, etc. 5. In HDFN due to ABO incompatibility, the immunizing stimulus is unknown and is not detected during pregnancy. (Group O mothers with naturally occurring IgG anti-ab, anti-a and anti-b. III. Effects of HDFN A. Anemia - Develops when hematopoiesis cannot keep pace with red cell destruction. 1. The greatest concern is in the fetus and in the first 24 hours after birth. 2. Erythroblastosis fetalis Red cell destruction and resulting anemia stimulates erythropoiesis in the fetus. Immature (nucleated) red cells are released into circulation. Extramedullary hematopoiesis leads to enlargement of the liver and spleen. 3. Hydrops fetalis - cardiac failure with generalized edema 4. Can range from subclinical to profound anemia causing stillbirth or neonatal death. B. Bilirubinemia - By-product of red cell degradation 1. Glucuronyl transferase, produced by the liver, conjugates indirect bilirubin to direct bilirubin, which is excreted 2. In fetus, bilirubin crosses the placenta and is conjugated and excreted by mother. In neonate, ability to conjugate and excrete bilirubin is not well developed 3. Kernicterus - high levels (>18 mg/dl) of unconjugated bilirubin accumulates in the fatty tissue of the brain, causing irreversible brain damage 4. Greatest concern in neonate; premature at higher risk than full term CLS 422 Clinical Immunohematology I Page 2 of 10

3 IV. Prenatal Testing and Treatment -testing performed on the mother to help assess the risk of and classify the cause of HDFN. -3 categories: Rh (due to anti-d), ABO, and other (includes other Rh antibodies). A. Serologic testing 1. ABO and Rh type -weak D testing is not required 2. Antibody screen B. Ultrasound a. Preferably done at the initial OB appointment. May be repeated before administration of antenatal Rh Immune Globulin (RhIG) or during the third trimester, if the woman has a history of transfusion or unexpected antibody. b. If an antibody is detected: 1) Identify 2) If IgG, type the father for pertinent antigen 3) If IgG, perform titer a) Serum is serially diluted and tested against appropriate cells - result is reciprocal of highest dilution that gives 1+ reaction. b) Parallel titrations with previous specimen must be done as a control c) Rise in titer of 2 tubes or greater, or increase in score of more than 10 points, may indicate baby is affected. 1. Non invasive, so lower risk to mother and fetus. 2. Allows overall monitoring of fetal wellness and disease progression. 3. Able to detect thickening of placenta, fetal liver enlargement, and hydrops fetalis (indications of severe anemia). 4. More recently, Doppler ultrasound is being used to determine the peak systolic velocity of the middle cerebral artery (MCA PSV) in the fetus. When the fetus begins to experience anemia the MCA PSV increases. This is also useful in monitoring the results of intrauterine transfusion. CLS 422 Clinical Immunohematology I Page 3 of 10

4 C. Amniocentesis 1. Level of bilirubin pigment is measured at an optical density at 450 nm a. Value is plotted on a Liley graph against age of gestation to determine fetal risk. b. Diagnostic procedure used to assess severity of HDFN in utero 2. Amniocytes may be harvested and tested for genes that control expression of the target antigen. 3. Risk of procedure causing a feto-maternal hemorrhage, which can lead to sensitization. Rh negative women should receive RhIG following procedure. D. Percutaneous umbilical cord blood sampling (PUBS) or cordiocentesis 1. Allows fetal specimen to be obtained for Hct, reticulocyte count, ABO/Rh, antigen typing and DAT determinations. 2. Creates access to fetal circulation if intrauterine transfusion necessary. 3. Risk of feto-maternal hemorrhage. Treat Rh negative women with RhIG. E. Intrauterine transfusion of fetus 1. Done to correct severe anemia and prevent death when premature delivery is not advisable. 2. Blood selection a. O Rh Negative b. Compatible with antibodies in mother s serum c. Fresh d. CMV negative e. Irradiated to prevent GVH disease CLS 422 Clinical Immunohematology I Page 4 of 10

5 f. Negative for Hgb. S - red cells from donors heterozygous for Hgb. S gene undergo sickling if exposed to extremely low oxygen tensions. The transfusion situation in which this may happen is a severely hypoxic or acidotic recipient whose blood contains few cells with high enough normal Hgb. concentration. An intrauterine or exchange transfusion in a severely ill infant could establish these conditions g. May be prepared to a certain Hct. 3. Transfused into the fetal abdominal cavity or umbilical vein. a. Cells are absorbed by lymphatic channels draining the peritoneal cavity. b. Usual dose is cc per transfusion. May need repeated transfusions. c. May complicate ABO/Rh determinations on infant following delivery. V. Postnatal Testing and Treatment A. Cord blood studies 1. Cord blood hemoglobin a. Most important index of severity b. Heel stick values not as accurate because of blood the baby receives from the placenta before the cord is cut 2. Cord blood bilirubin a. Bili level rises as RBCs destroyed b. Cord levels >4.0 mg/dl and heel stick levels >20 mg/dl are associated with kernicterus c. Non-immunologic causes of hyperbilirubinemia: 1) G-6-PD deficiency 2) Hereditary spherocytosis 3) Drug therapy 4) Infections 5) Reabsorption of large hematomas CLS 422 Clinical Immunohematology I Page 5 of 10

6 3. Serologic testing - Cord blood from infants born to all Rh negative mothers and mothers with a history of clinically significant antibodies must be tested. Many institutions also test cord blood from infants born to Group O mothers. a. ABO and Rh type 1) A and B antigens may not be fully developed, therefore anti-a,b is often included in testing. 2) Reverse type is not performed 3) Possible contamination with Wharton s jelly 4) Weak D testing is necessary, if the mother is Rh negative b. DAT 1) Most important serologic test in diagnosis of HDFN 2) Detects in vivo sensitization of baby s red cells 3) Strength of reaction does not correlate with severity of disease 4) Can help differentiate between immune and physiologic jaundice c. Elution If DAT is positive with anti-igg AHG. 1) Removes antibody from the baby s cells so it can be identified 2) Test eluate with A and B cells and group O screen cells B. Management of HDFN 1. Phototherapy using blue to blue-green light (430- to 490-nm band) to breakdown bilirubin. 2. Small volume transfusion a. Used to correct anemia of prematurity or iatrogenic blood loss. b. May utilize quad packs, in order to maximize unit use while minimizing donor exposure. (small aliquot bags that have been joined to the RBC unit under sterile conditions that maintain a closed system) 3. Exchange transfusion - originally used almost exclusively for hemolytic disease of the fetus and newborn, has recently been advocated as adjunctive therapy for a variety of life-threatening diseases affecting newborns including respiratory distress, CLS 422 Clinical Immunohematology I Page 6 of 10

7 hyperbilirubinemia, disseminated intravascular coagulation (DIC) and sepsis a. Four main purposes in HDFN: 1) Remove bilirubin 2) Remove sensitized RBC 3) Remove free antibody 4) Supply RBCs to correct anemia b. Criteria 1) Rate of rise of bilirubin level. Lower levels more significant in preemie 2) Degree of anemia c. Selection of blood 1) Negative for antigens corresponding to mother s antibody 2) ABO/Rh compatible with infant s blood group 3) Negative for hemoglobin S 4) CMV negative 5) Freshest 6) Irradiated 7) Prepared to a specified hct 4. Compatibility testing a. If the initial antibody screen using the infant s plasma is negative, no additional compatibility testing is necessary for that admission, until the infant reaches 4 months of age. b. If the screen is positive, donor units must be antigen negative and/or antiglobulin crossmatch compatible, until the maternal antibody is no longer detected in the neonate s plasma. c. Specimen of choice for crossmatching is mother s serum 1) More abundant specimen 2) Higher titer of antibody 3) Available before delivery d. Baby s serum or eluate may be used for crossmatch (much of mother s antibody may be coating the infant s cells). e. If a non-group O infant is receiving non-group O red cell products, the infant s plasma must be tested for the presence of maternal anti-a, -B, or AB, using an antiglobulin test. CLS 422 Clinical Immunohematology I Page 7 of 10

8 VI. Prevention of Rh HDFN (Rh Immune Globulin - RhIG) A. Action - RhIG is purified human anti-d gamma globulin. It suppresses the formation of immune anti-d when given to Rh neg mothers of Rh positive babies by blocking the D antigen sites on baby s red cells with anti-d from the RhIG. With those antigen sites masked, the mother is not stimulated to produce immune anti-d. The ½ life of RhIG is about 23 days. B. Criteria for candidates for RhIG 1. Mother is Rh negative 2. Baby is Rh positive, weak D positive or Rh unknown 3. Mother has not already formed anti-d (immune anti-d) a. Active, immune anti-d may have IgM form that is reactive at the immediate spin phase as well as 37 o C, and may be high titered. b. Passive anti-d from RhIG is IgG only, and usually does not titer over 4. C. Administration 1. Ante partum a. At 28 weeks gestation to protect against minute leaks of fetal blood into maternal circulation. b. Following invasive procedures, such as amniocentesis, or following ectopic pregnancy or trauma. 2. Postpartum a. Within 72 hours of delivery of Rh positive infant b. Following abortion 3. Dose a. 300ug dose is sufficient for protection against 15 ml of Rh pos packed red cells (30 ml of fetal whole blood) b. Given intramuscularly (typically in the buttocks) CLS 422 Clinical Immunohematology I Page 8 of 10

9 c. Does not transmit HIV or hepatitis D. Detection of feto-maternal bleeding 1. Feto-maternal hemorrhage (FMH) screen - qualitative test a. Ideal specimen is an EDTA tube collected 1 hour after delivery. b. Detects large feto-maternal hemorrhage c. Uses rosette procedure of enzyme treated Rh positive indicator cells d. Screen is not accurate for weak D positive infants - false negative Screen is not accurate for weak D positive mothers - false positive Screen is not accurate if infant has a positive DAT (cells already coated with antibody) - false negative e. If FMH screen is positive (or cannot be accurately performed) a quantitative test is performed.. 2. Kleihauer-Betke acid elution - quantitative test for feto-maternal hemorrhage a. Principle - adult red cells containing Hgb A are soluble in an acid buffer, while fetal red cells containing Hgb F are not soluble b. Calculations: # fetal cells / # total cells counted x maternal blood volume (often estimated at 5000 ml) = mls fetal whole blood mls of fetal whole blood / 30 = # of vials of RhIG to administer - If # of vials is a fraction, then <.5 round down, >.5 round up. - It is usually recommended that one additional vial beyond what is calculated be administered (overkill). E. Other applications for Rh immune globulin: 1. After infusion of Rh positive platelets. 2. After accidental infusion of Rh positive red cells. 3. May be given IV for these cases. CLS 422 Clinical Immunohematology I Page 9 of 10

10 VII. ABO HDFN - Most common form of HDFN now, due to RhIG preventing Rh HDFN. A. Etiology 1. Occurs when mother has antibody directed against baby s ABO antigens 2. Usually occurs in group O mothers - IgG anti-ab crosses placenta and attaches to red cells of A or B baby (May also be IgG anti-a or Anti-B) 3. Usually mild disease - antibody may be neutralized by ABO antigens in tissues and secretions % of pregnancies are ABO incompatible; however ABO HDFN only occurs in 0.7-2%. B. Serology 1. DAT - usually weakly positive or may be negative 2. Eluate - anti-a, anti-b, or anti-a,b CLS 422 Clinical Immunohematology I Page 10 of 10

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