Conservative Management of Alloimmune Hemolysis and Cholestasis With Extreme Laboratory Abnormalities

The first patient presented to our care at day-of-life (DOL) 21 with a normocytic anemia and conjugated hyperbilirubinemia (Fig 1 A and B). His medical history was pertinent for HDFN secondary to anti-E, anti-c, and anti-Fy(a) alloimmunization caused by fetal-maternal hemorrhage during a previous pregnancy. He required 4 in utero transfusions (IUTs). He was delivered at 37 weeks’ gestational age at an outside institution. At 7.5 hours of life, his laboratory evaluation was notable for hemoglobin (Hb) of 10.9 g/dL, total bilirubin of 11.1 mg/dL, and direct bilirubin of 5.3 mg/dL. He was started on phototherapy and received 2 doses of intravenous immune globulin (IVIg) on DOL 0 and 1 for alloimmune-mediated hemolysis. Abdominal ultrasound revealed trace ascites but otherwise normal anatomy. Phototherapy was discontinued on DOL 3. He was discharged on DOL 5, and repeat laboratory evaluation on DOL 7 revealed total bilirubin of 16.5 mg/dL and direct bilirubin of 15.0 mg/dL. A hepatobiliary iminodiacetic acid scan was unremarkable, and he was initiated on off-label ursodiol for the treatment of cholestatic liver disease. On DOL 21, testing was notable for Hb of 6.6 g/dL, reticulocyte count at 12.9%, total bilirubin of 16.5 mg/dL, and conjugated bilirubin of 13.8 mg/dL. He was admitted to our hospital for further evaluation. He was breastfeeding well with yellow stools and urine. His examination was notable for jaundice to the level of his navel, scleral icterus, a systolic ejection murmur, and hepatic edge 2 cm below the right-costal margin. Additional laboratory evaluation was notable for alanine aminotransferase (ALT) 642 U/L (12.8-fold upper limit of normal [ULN]), aspartate aminotransferase (AST) 994 U/L (14-fold ULN), alkaline phosphatase 818 U/L (2.7-fold ULN), and ferritin 21 000 ng/mL; he had a normal-for-age albumin level and coagulation tests. His α-fetoprotein was 26 200 ng/mL and repeat ferritin on DOL 22 was 24 700 ng/mL. His blood type was A/Rh+ with anti-E and anti-c antibodies, with only the former present in his RBC eluate suggesting that anti-E antibody was primarily responsible for his ongoing hemolysis. Genetic, metabolic, and infectious etiologies were excluded on the basis of extensive workup that included a 23-gene cholestasis panel, chest and abdominal radiographs, sweat chloride test, serum pyruvate, plasma amino acids, urine organic and bile acids, urine-reducing substances, and polymerase chain reaction (PCR) testing for herpes simplex virus, hepatitis B and C, Epstein-Barr virus, and cytomegalovirus (CMV). Hemophagocytic lymphohistiocytosis was considered unlikely because the patient did not demonstrate fever, splenomegaly, hypofibrinogenemia, or cytopenias other than anemia; no additional testing for this etiology was obtained. The patient received 15 mL/kg of packed red blood cells (pRBCs), and on the basis of his clinical condition, he was discharged with close outpatient follow-up. His laboratory studies improved within the next 6 months (Fig 1A).

The second patient presented to our institution at DOL 27 with worsening direct hyperbilirubinemia (Fig 1C). Her medical history was notable for HDFN secondary to anti-D, anti-C, and anti-S alloimmunization, the former being notable because her mother did not receive Rho(D) immune globulin during a previous pregnancy. She received 7 IUTs between 19 weeks’ gestation and delivery at 35.5 weeks’ gestational age. At 24 hours of life, she developed jaundice and was initiated on phototherapy. She received 2 doses of IVIg. She required 10 days of total parenteral nutrition because of poor feeding and hypoglycemia. She was discharged from the hospital on DOL 13, and total bilirubin at that time was 1.8 mg/dL. She was admitted on DOL 23 to an outside institution with worsening jaundice, an Hb of 7.3 g/dL, total bilirubin of 8 mg/dL, and direct bilirubin of 2.8 gm/dL. She received 35 mL/kg pRBC transfusion and was discharged from the hospital. She was admitted to our institution for worsening laboratory studies that peaked on DOL 36 with ALT 1406 U/L (33.5-fold ULN), AST 937 U/L (14.6-fold ULN), total bilirubin of 9.9 mg/dL, direct bilirubin of 6.5 mg/dL, Hb of 13.7 g/dL, and reticulocyte count at 0.1%, (Fig 1 C and D). Her blood type was A/Rh+ with anti-D and anti-C antibodies in her RBC eluate. Because of her worsening conjugated hyperbilirubinemia, genetic, metabolic, and infectious etiologies were excluded on the basis of a workup that included α-1-antitrypsin genotyping, sweat chloride testing, chest and abdominal radiograph, serum lactate and pyruvate, acylcarnitine profile, urine organic and bile acids, urine-reducing substances, and PCR testing for hepatitis B, hepatitis C, Epstein-Barr virus, and CMV. Her abdominal ultrasound and hepatobiliary iminodiacetic acid scan were normal. Liver biopsy was performed on DOL 42, which revealed neonatal hepatitis pattern of injury with focal mild portal fibrosis, mild pericentral sinusoidal fibrosis, and marked reticuloendothelial (3+) and hepatocellular (2+) iron deposition (Fig 2). Biopsy results were not consistent with hemophagocytic lymphohistiocytosis. She was started on off-label ursodiol on DOL 44. Ferritin obtained at DOL 55 was 5204 ng/mL. The patient demonstrated continued decline in Hb with reticulocytopenia at DOL 70 and was started on epoetin α to minimize additional transfusions and iron loading. Parvovirus and repeat CMV PCR results were negative. Over time, her laboratory studies improved (Fig 1 C and D).

The incidence of cholestasis in infants with HDFN is ∼10%,^6,7,9  with anti-D alloimmunization and IUTs as identified risk factors.⁹  HDFN-associated cholestasis has rarely been reported with alloimmunization against ABO antigens^4,10,12,20  or other RBC antigens,^4,9,24  as was as observed in our first case. Several biological mechanisms have been proposed to contribute to cholestasis. Historically, inspissated bile syndrome was suggested because of direct occlusion of the bile collecting system by the large flux of bilirubin in dehydrated bile resulting from the hemolysis.^5–8  More recently, direct-iron toxicity on the fetal or infant liver has been implicated, with liver biopsies revealing iron deposition in hepatocytes,^9,13–17  as was observed in our second case. Several studies have revealed that infants with HDFN, even without liver disease, have high plasma ferritin levels suggestive of iron overload, which is worsened in patients who received IUT.^25–28  The fetal or newborn liver may be uniquely sensitive to iron toxicity because of the immaturity of antioxidant systems,²⁷  which when combined with hemolysis¹⁹  and pRBC transfusions can result in iron overload and potentially hepatotoxicity.

Infants with HDFN-associated cholestasis, especially with extreme laboratory abnormalities, pose a diagnostic and therapeutic dilemma for pediatricians, with authors of case reports and series reporting a broad spectrum of diagnostic evaluations and a variety of therapeutic interventions beyond phototherapy and pRBC support (Table 1). For >3 decades, authors have urged investigational restraint for this syndrome,²⁹  which Smits-Wintjens et al⁹  recently reiterated, suggesting that a full laboratory and radiographic evaluation of etiologies of cholestasis is not indicated in patients with HDFN treated with at least 1 IUT who are without additional medical concerns. In our 2 cases, the extreme laboratory abnormalities motivated a more extensive evaluation for both patients. Retrospectively, our experience further supports judicious evaluation because both cases had HDFN-associated cholestasis, IUT, and varying degrees of hepatocellular injury that triggered broad evaluations (including liver biopsy) that were unrevealing for alternative diagnoses. Case 1 and others^{9,13,16,17,21}  highlight that extreme hyperferritinemia is consistent with HDFN-associated cholestasis; likewise, case 2 and others¹⁸  indicate that liver transaminases even >1000 U/mL may also be part of this syndrome.

Thus, in a well-appearing infant who has received IUT for HDFN, we believe it is reasonable to document the presence of a gallbladder and bile ducts with minimally invasive and low-cost imaging such as ultrasound. Risk-based assessment of transfusion-transmitted infections may be warranted depending on maternal testing and risk factors of the local blood supply. Indeed, a recent case report revealed transfusion-associated CMV in an infant with HDFN-associated cholestasis, which likely exacerbated the clinical picture.²¹  However, our experience suggests that practitioners can consider deferring other costly and invasive evaluations.

Authors of several case reports have described iron chelation with deferoxamine in infants with HDFN-associated cholestasis and hyperferritinemia (Table 1). Although authors of one case reported marked coagulopathy²³  and another hypoalbuminemia,¹⁴  the majority did not describe problems with liver synthetic dysfunction.^9,15–17  Given that our patients were without synthetic liver dysfunction and clinically stable, we deferred initiation of chelation therapy with deferoxamine; subcutaneous deferoxamine infusions are associated with infusion-site reactions, allergic reactions including anaphylaxis, and cytopenias and renal dysfunction. Our cases highlight that iron chelation is not always necessary for HDFN-associated cholestasis and hyperferritinemia, even with biopsy-proven hepatic iron overload. This syndrome can self-resolve in a few months’ time (Fig 1).

Ursodiol is a naturally occurring bile acid therapeutically used to increase bile flow in cholestatic liver disease³⁰ ; although not Food and Drug Administration approved in pediatrics, it has been used as an adjuvant therapy in infants with cholestatic disorders,^30–32  including in HDFN-associated cholestasis (Table 1). However, authors of recent work have raised alarms about the use of ursodiol in infants, including an association with increased mortality.³³ 

Both of our patients and many of the published cases (Table 1) received IVIg. IVIg remains a recommended intervention for HDFN and hyperbilirubinemia,³⁴  although there are limited data supporting its efficacy for HDFN.¹  IVIg is highly efficacious in the treatment of gestational alloimmune liver disease, which unlike in our 2 cases, typically presents with acute liver failure.³⁵  Postnatal pRBC transfusion benefit must be weighed against the associated iron loading with each treatment. Our second patient demonstrated increasing cholestasis after pRBC transfusion on DOL 30; thus, we initiated erythropoietin therapy, as has been used in a number of other cases (Table 1). Concomitant iron supplementation should likely be avoided.³⁶ 

Cholestasis with hyperferritinemia has been described rarely in patients with HDFN, and we present 2 additional cases. Our findings help define the spectrum of laboratory abnormalities that are consistent with cholestasis associated with HDFN requiring IUT. In future work, researchers should focus on developing evidenced-based diagnostic and therapeutic approaches to these patients.

ALT

alanine aminotransferase

AST

aspartate aminotransferase

CMV

cytomegalovirus

DOL

day of life

Hb

hemoglobin

HDFN

hemolytic disease of the fetus or newborn

IUT

in utero transfusion

IVIg

intravenous immune globulin

PCR

polymerase chain reaction

pRBC

packed red blood cell

RBC

red blood cell

ULN

upper limit of normal