A Mysterious Case of Persistent Hemolytic Disease of the Fetus/Newborn Blood Centers of the Pacific BloodSource United Blood Services A Mysterious Case of Persistent Hemolytic Disease of the Fetus/Newborn Marissa Li, MD Medical Director United Blood Services mli@bloodsystems.org

Agenda Case report Breastfeeding and breast milk Statistics Professional organization recommendations Composition and benefits Hemolytic disease of the fetus/newborn (HDFN) Etiology HDFN effects on fetus/neonate Prevention Diagnosis Management

Case Report

Mother’s clinical history 32 year old G3P3 O negative female with anti-D, anti-G and recurrent history of HDFN First pregnancy resulted in a near term intrauterine fetal demise Second pregnancy resulted in a preterm, severely anemic child requiring postnatal exchange transfusions Current pregnancy complicated by evidence of intrauterine hydrops fetalis due to high titer anti-D She other underwent two intrauterine transfusions (O neg RBC) in June 2015, the first when fetal Hb was 2.0 g/dL and the second when the fetal Hb was 3.8 g/dL Despite two intrauterine transfusions fetus continued to display signs of distress Minimal improvement of MCA doppler result and persistent fluid accumulation She delivered a 1,740 gram, 28 week gestation preterm male on 06/21/2015 via Cesarean section

Mother and neonate’s serologic findings O neg Anti-D in plasma (high titer, not further quantified) Anti-D in breast milk (4+) Neonate B pos Anti-D in plasma (3+) Positive IgG DAT (3+) Eluate not performed

Neonate’s clinical history Date Labs Exchange or simple transfusions Additional treatment for HDFN Nutrition 06/21/15 (day one of life) Hb 4.8 g/dL 15.9 g/dL T-bili 4.7 mg/dL; D-bili 0.6 mg/dL; I-bili 4.1 mg/dL Normal LFT Double volume exchange transfusion (O neg RBC) Phototherapy IVIg NPO, TPN 06/26/15 Hb 10.5 g/dL 13.4 g/dL T-bili 8.0 mg/dL Two simple transfusions (25 mL and 30 mL O neg RBC) 06/29/15 Hb 10.9 g/dL 16 g/dL 07/02/15 Started maternal breast milk feeding 07/08/15 Hb 11.5 g/dL 14.8 g/dL T-bili 8.8 mg/dL 6.6 mg/dL Discontinued oral feeding for exchange transfusion and concerns for NEC 07/09/15 Identified anti-D in breast milk 07/17/15 Hb 8.4 g/dL 11.7 g/dL Anemia secondary to bloody stool & iatrogenic causes Simple transfusion (40 mL O neg RBC) 07/20/15 Stable Hb Started donor breast milk feeding 11/02/15 (discharge) Hb 11.5 g/dL T-bili 1.0 mg/dL Infant formula started at 1month and 15 days of age till discharge

Breast milk & breastfeeding

Trivia question Which of the following is the most abundant component of the breast milk Protein Carbohydrate Fat Water Vitamins

Breastfeeding statistics ~38% of infants are exclusively breastfed in the world In the US 75% of infants initiate breastfeeding from birth 67% of infants rely on infant formula for some portion of their nutrient by 3 months of age 43% of mothers breastfeed for the first 6 months but only 13% breastfeed exclusively Martin CR, et al. Nutrients 2016

Professional organization view points on breastfeeding WHO Recommends exclusive breastfeeding for first 6 months of life American Academy of Pediatrics Recommends breastfeeding for ≥ 12 months Academy of Nutrition and Dietetics Recommends exclusive breastfeeding for first 6 months of life and complement with food from 6 months until ≥ 12 months of life Martin CR, et al. Nutrients 2016

Breast milk composition Water: 87% Carbohydrates: 7% Human milk oligosaccharides are anti-microbial and protect against gut pathogens such as Salmonella, Listeria and Campylobacter Fat: 3.8% Most important composition of milk by supplying energy and helping with CNS development Protein: 1% Vitamins Minerals Digestive enzymes Hormones Immune cells Bioactive molecules Martin CR, et al. Nutrients 2016

Immune composition of breast milk Human milk contains immune cells and antibodies protective against numerous pathogens the mother has been exposed to throughout her lifetime Antibody level falls throughout lactation but the amount of immunoglobulins received by the child remains unchanged due to increase milk intake Palmeira P and Carneiro-Sampaio, M. Rev Assoc Med Bras 2016

Immunoglobulin compositions in breast milk All classes of immunoglobulins are found in milk (IgA > IgM > IgG) IgA/SIgA (secretory IgA) is the most important in relation to concentration and biological properties Make up 80-90% of total immunoglobulins in human milk Only 10% is absorbed in the intestines and transferred to the bloodstream Defend mucous membranes and prevent entry of microorganisms into tissues Anti-inflammatory IgM Reactive with viruses and bacteria Protect mucosal surfaces IgG Neutralizing and opsonizing activity Activate complement system and antibody-dependent cytotoxicity Palmeira P and Carneiro-Sampaio, M. Rev Assoc Med Bras 2016

Benefits of breast milk Protect against infections such as otitis media, upper and lower respiratory track infections, gastroenteritis, sepsis and meningitis Convenient, inexpensive, provide bonding experience for mother and infant Breast milk oligosaccharides and antibodies can help protect against diseases commonly afflicting premature and underweight infants Sepsis, meningitis and necrotizing enterocolitis Martin CR, et al. Nutrients 2016 Palmeira P and Carneiro-Sampaio, M. Rev Assoc Med Bras 2016

HDFN Overview

Trivia question Which maternal antibody(ies) is the most common cause of HDFN Anti-E Anti-K ABO antibodies Anti-D Anti-c

Etiology HDFN is caused by maternal red cell antibodies Naturally occurring ABO antibodies & alloantibodies developed through prior pregnancies, transfusion or transplant Rate of hemolysis and severity of disease is determined by the following factors Antibody class IgM: not transported into fetal circulation IgG subclass IgG is actively transported across the placenta (class 1 & 3 are most efficiently transported) Amount of antibody Number of antigenic sites on the fetal/neonatal red cells AABB Technical Manual 18th Edition

HDFN effects on fetus & infants Fetus affected by HDFN develop Anemia and compensatory erythropoiesis Anti-K also cause erythroid progenitor cell destruction in the marrow which prevents compensatory erythropoiesis Hyperbilirubinemia In utero: maternal liver metabolizes the bilirubin Postnatal: infant hepatic enzyme insufficient at metabolizing elevated bilirubin, this may lead to kernicterus Organomegaly Hydrops fetalis Fetal skin/organ edema and eventually intrauterine death De Haas, M et al. Hemolytic disease of the fetus and newborn. Vox Sanguinis 2015

De Haas, M et al. Hemolytic disease of the fetus and newborn De Haas, M et al. Hemolytic disease of the fetus and newborn. Vox Sanguinis 2015

Prevention Prenatal maternal ABO/Rh typing and antibody screen/ID If mother is Rh(D) negative then initiate RhIg prophylaxis at 28 weeks 92% of women develop anti-D ≥ 28 week gestation Decrease the risk of Rh(D) alloimmunization from 16% to <0.1% If mother’s antibody screen is positive with specific alloantibody If alloantibody is known to potentially cause HDFN Close monitoring of fetus for signs of anemia Close monitoring of maternal alloantibody titer If alloantibody is NOT known to cause HDFN May choose to repeat antibody screen/ID during pregnancy to monitor for change in antibody class or new antibody development AABB Technical Manual 18th Edition

Diagnosis Diagnosis of HDFN can be made by identifying clinically significant alloantibody during pregnancy Antibody screen/ID and titer as appropriate Consider non-invasive fetal blood group typing with cell free fetal DNA from maternal plasma if available OR Changes in fetal/neonatal well being Intrauterine: decrease fetal movement, sudden death Postnatal: neonatal jaundice and anemia AABB Technical Manual 18th Edition De Haas, M et al. Hemolytic disease of the fetus and newborn. Vox Sanguinis 2015

Impact of antibody specificity on severity of HDFN De Haas, M et al. Hemolytic disease of the fetus and newborn. Vox Sanguinis 2015

Management Prenatal management options Postnatal management options Measure middle cerebral artery peak systolic velocity to detect fetal anemia Moderate to severe anemia: velocity >1.5 multiples of the median (MoM) Intrauterine transfusions Pros Correct anemia Reduce the burden of antigen positive red blood cells Reduce the amount of maternal alloantibody Cons Further alloimmunization in 25% of mothers despite matching for Rh/K Postnatal management options Phototherapy Reduce unconjungated bilirubin and risk for kernicterus Exchange transfusions Reduce unconjungated bilirubin Remove maternal alloantibodies Correct anemia Simple transfusions De Haas, M et al. Hemolytic disease of the fetus and newborn. Vox Sanguinis 2015

Case conclusion

Differential diagnosis of HDFN in this neonate Anti-D associated HDFN Anti-D from maternal breast milk Maternal breast milk contained high concentration of anti-D Maternal breast milk cessation is well correlated with neonate’s Hb recovery Late hyporegenerative anemia caused by ongoing intramedullary hemolysis of Rh positive neonatal erythroid precursor and/or marrow suppression by the high titer anti-D Ongoing clinical evidence of hemolysis (i.e. elevated bilirubin and persistent anemia) in the presence of negative DAT in the neonate Anti-D from maternal circulation GI track absorption of immunoglobulin is variable High titer anti-D from maternal circulation may have persisted despite 3 double volume exchange transfusions and numerous simple transfusions ABO associated HDFN (Mom: O neg; Neonate: B pos) Severity of hemolysis despite transfusing O neg RBC History of anti-D mediated HDFN High titer anti-D in current pregnancy a study of neonates breast fed by mothers with immune thrombocytopenic purpura demonstrated that sufficient amount of IgA class anti-platelet antibodies can be effectively absorbed by neonatal GI track leading to persistent thrombocytopenia4. Moreover, a murine model of passively acquired maternal alloantibody (IgA and IgG) via breast milk suggests that perhaps the intestinal absorbance of immunoglobulin in human neonates may be more efficient than previously reported3.

The End……..