refractory cholestasis presenting as cholangiolitis in an rh (e)-incompatible neonate

$: Refractory cholestasis presenting as cholangiolitis in an Rh (E)-incompatible neonate$
INSTRUCTIVE CASE

Refractory cholestasis presenting as cholangiolitis in an Rh(E)-incompatible neonatejpc_1874 126..131

Ryusuke Taba,1 Masaru Yamakawa,1 Chisato Miyakoshi1 and Yukihiro Imai2

Departments of 1Pediatrics and 2Clinical Pathology, Kobe City Medical Center General Hospital, Kobe, Japan

Abstract: Cholestasis in neonates is infrequently associated with Rh isoimmunization, and usually resolves within a month. The suggestedpathophysiology is inspissated bile and hepatocellular damage. We report a rare case of refractory cholestasis presenting with cholangiolitis ina newborn with anti-E isoimmunisation. The cholangiolitis was disclosed by immunohistochemical investigation of conjugated hyperbilirubi-naemia and by liver biopsy, which showed a number of CD8+ lymphocytes within the portal tract damaging the interlobular bile duct. Bilirubinlevels dramatically decreased after 14-day corticosteroid therapy (prednisolone, 2 mg/kg/day) implying that the cause of cholestasis could beimmune-mediated cholangiolitis.

Key words: conjugated bilirubin; hemolytic disease of the newborn; cholestasis; steroid.

Introduction

Conjugated hyperbilirubinaemia is an uncommon presentingfeature of haemolytic disease of the newborn (HDN).1–3

Cholestasis in HDN is usually mild and resolves within amonth.1,2,4 The suggested pathogenesis of cholestasis is bileinspissation caused by excessive bilirubin load,1 functionaland/or anatomical hepatocellular damage directly produced byunconjugated hyperbilirubinaemia and/or secondary to heart

failure or anaemia,1,2 and liver iron overload induced bymultiple intra-uterine blood transfusions and/or exchangetransfusions (ETs).3

Biopsies from cholestatic livers of newborns with haemolyticdisease are reported to show non-specific changes, includingliver cell necrosis, giant cell regeneration, bile plug formationand hepatic fibrosis.1,2

We report an unusual case of early-onset refractory intrahe-patic cholestasis presenting as cholangiolitis complicated withHDN caused by anti-E.

Method

This was a case report and immunohistochemical investigation.The institutional ethical committee approved publication afterobtaining parental informed consent.

Immunohistochemistry: immunohistochemical investigationwas performed using an Envision kit (Dako Japan, Kyoto,Japan). Primary antibodies used in this case were cytokeratin (X50; DAKO) and CD8 (prediluted; Nichirei Biosciences Inc.).

Case Report

A male infant (birth length, 50.2 cm; birthweight, 3244 g; Apgarscores, 7 (at 1 min) and 8 (at 5 min) ) was born at 40 weeksgestation (estimated by last menstrual period) via spontaneousvaginal delivery with meconium staining of the skin. Hismother, a healthy 29-year-old primigravida, chose not to acceptroutine prenatal care. She previously received a blood transfu-sion after a traffic accident at age 25. Her blood group was AB,and antibody screen revealed increased Rh anti-E antibody titre.She was typed as Rh(D) positive, C positive, E negative, cnegative and e positive. Her serology for syphilis, human immu-nodeficiency virus, hepatitis B and hepatitis C was negative.His father (a Japanese male) was typed as Rh (D) positive,

Key Points

1 Cholestasis is an uncommon presenting feature of haemolyticdisease of the newborn (HDN) and resolves within a month.

2 The suggested pathogenesis of cholestasis is bile inspissationcaused by excessive bilirubin load,1 functional and/or anato-mical hepatocellular damage directly produced by unconju-gated hyperbilirubinemia, and/or secondary to heart failure oranaemia1,2 and liver iron overload induced by multiple intra-uterine blood transfusions and/or exchange transfusions.3

3 This case of refractory intrahepatic cholestasis was in anewborn presenting with cholangiolitis disclosed by immuno-histochemical investigation showing a number of CD8+ lym-phocytes within the portal tract, damage to the interlobularbile duct, and anti-E isoimmunisation.

4 Bilirubin levels decreased dramatically after 14 days of corti-costeroid therapy (methylprednisolone, 2 mg/kg/day) implicat-ing an inflammatory process (e.g., cholangiolitis) as the causeof the cholestasis. Corticosteroids might be adjunctive treat-ment for refractory cholestasis in HDN.

Correspondence: Dr Ryusuke Taba, Department of Paediatrics, Kobe CityGeneral Hospital, 4-6 Minatojima-nakamachi, Chuo-ku, Kobe 650-0046,Japan. Fax: + 81 78 302 2487; email: [email protected]

Accepted for publication 25 March 2010.

doi:10.1111/j.1440-1754.2010.01874.x

Journal of Paediatrics and Child Health 48 (2012) E126–E131© 2010 The Authors

Journal of Paediatrics and Child Health © 2010 Paediatrics and Child Health Division (Royal Australasian College of Physicians)

E126

C negative, E positive, c positive and e negative. There was nofamily history of either early neonatal death or neonatal severejaundice. At birth, the child needed oxygen (FiO2 25%) by nasalcannula and had jaundice. The post-ductal SpO2 was 94% withno respiratory distress a few minutes after initiating supplemen-tal oxygen. His haemoglobin was 7.2 g/dL; nucleated red bloodcells, 1.85 ¥ 1012/L; corrected white blood cell, 15.4 ¥ 109 /L;haematocrit, 22.8%; platelet count, 233 ¥ 109/L; total serumbilirubin (TSB), 201.8 mmol/L; albumin, 34 g/L; prothrombinand active partial thromboplastin time (Table 1.), normal; bloodgroup, B Rh positive; direct antiglobulin test, positive. His initialconjugated bilirubin (CB) level and reticulocyte count were notobtained. At 3 h after birth, human intravenous immune globu-lin (IVIg; 1 g/kg) was administered along with intensive photo-therapy. At 10 h after birth, his haemoglobin was 7.1 g/dL;nucleated red blood cells, 1.52 ¥ 1012/L; haematocrit, 22.5%;

reticulocyte count (relative to RBC), 11.6%; TSB/CB, 270.2/164.2 mmol/L; albumin, 3.5 g/L; ferritin, 135 mg/dL (Table 1);alpha-1-antitrypsin, 2.3 g/L.

Viral polymerase chain reactions for cytomegalovirus, herpessimplex virus, rubella, human herpes virus 6, varicella zosterand parvovirus from his blood, liver, and placenta as well asviral isolation for echo, coxsackie virus and adenovirus from hisstool and urine were all negative. Serology for hepatitis B and Cwere negative in the infant.

Screening by tandem mass spectrometry was negative in thenewborn on day 5. Plasma amino acids on day 7 and urineorganic acids on day 5 were at normal levels. Blood, urine,nasopharyngeal aspirates and cerebrospinal fluid culturesremained sterile. The screen for glucose-6-phosphate dehydro-genase deficiency using the fluorescent spot test was negative.Direct gene sequencing found no UDP-glucuronosyltransferase1A1 gene mutation.

On the basis of TSB (338.6 mmol/L) at 24 h after birth(CB 202.1 mmol/L), he received double volume ET (160 mL/kg) with packed red blood cells (reconstituted in freshfrozen plasma with an adjusted haematocrit of 50%) viathe basilic vein. TSB/CB was reduced to 138.5/116.3 mmol/Lat 30 h. The phototherapy was halted within 10 h as the CBwas high. Ursodeoxycholic acid (UDCA; 20 mg/kg/day) andphenobarbital (PB; 5 mg/kg/day) were commenced on day2. TSB/CB peaked at 533.5/316.4 mmol/L on day 9. Doublevolume ET with RBC reconstituted as aforementioned wasrepeated. TSB/CB was reduced to 215.5/172.7 mmol/L onday 10. Abdominal ultrasound, performed twice a weekfrom birth until discharge, had demonstrated non-enlargedintrahepatic bile ducts with no gallstones. The cholestasis per-sisted (TSB/CB, 191.5/167.6 mmol/L to 350.6/304.4 mmol/L)for over a month. Operative cholangiography on day 32 foundan intact extrahepatic biliary tree with gall bladder. Liverbiopsy on day 32 showed moderate centrilobular cholestasis inhepatocytes and bile canaliculi accompanied by lymphocyticinfiltrates. Portal infiltrates were composed mainly of lympho-cytes, with occasional neutrophils but no eosinophils. Hepatitiswas absent. There was no bile ductular proliferation. Manyportal tracts contained destructed interlobular bile ducts withlymphocyte infiltration (Fig. 1a) at the peri-interlobular bileducts (cholangiolitis). Most of the infiltrated lymphocytes atthe periphery of the interlobular bile ducts were revealedas CD8+ lymphocytes (Fig. 1b), indicating immune-mediatedcholangiolitis. The infant was discharged from the neonatalintensive care unit on day 32 and received close follow-upat an intermediate care unit. Hepatobiliary scan on day 35showed no excretion into the small intestine (Fig. 2a). Bothbrain Magnetic Resonance Imaging (MRI) and auditory brain-stem responses (ABRs) conducted on day 30 were normal. Onthe basis of the pathological findings, immunosuppressivetreatment with intravenous methylprednisolone (2 mg/kg perday) was commenced on day 35. UDCA and PB (being inef-fective) were terminated. Elimination of the tracer was seenseven days later (day 42) (Fig. 2b). TSB/CB (350.6/304.4 mmol/L (Table 1.) at the start of methylprednisolonetreatment) fell to 115.1/83.8 mmol/L (Fig. 3) after 14 days ofthe intravenous methylprednisolone (2 mg/kg per day) andthen tapered over two months. There was no recurrence of

Table 1 Relevant investigations and the result of the liver biopsy

At birth 10 h Day 35 Normal range

WBC (¥109/L) 15.4 19.8 14.1 10–26

RBC (¥1012/L) 1.85 1.52 3.8 3.7–6.5

Hb (g/dL) 7.2 7.1 12.4 14.9–23.7

Ht (%) 22.8 22.5 49.3 47–75

Reti (%) N/A 11.6 4.5 3.0–6.9

Plt (¥109/L) 233 228 241 150–450

PT–INR 1.04 1.06 1.08 0.96–1.26

APTT (s) 35 31 32 24–36

Fibrinogen (g/L) N/A 1.92 2.06 1.10–3.04

Haptoglobin (mg/dL) N/A <10 N/A 43–180

TP (g/L) 56 53 51 56–85

Alb (g/L) 34 35 37 31.6–52.6

AST (U/L) 94 87 105 11–59

ALT (U/L) 19 31 189 4–30

GGT (U/L) N/A 204 843 50–150

TBA (U/L) N/A 256 286 <10

ALP (U/L) N/A 974 1403 190–963

LAP (U/L) N/A 387 562 2–310

LDH (U/L) 856 892 441 364–823

CK (U/L) 473 491 110 30–140

TSB (mmol/l) 201.8 270.2 350.6 1.7–302.7

CB (mmol/l) N/A 164.2 304.4 1.7–37.6

Iron (mmol/l) N/A 25.3 32.1 10–30

Ferritin (mg/dl) N/A 135 179 95–220

CRP (mg/dl) 0.4 0.2 0.4 0.1–1.1

N/A, not available.

Normal range: Neil Mclntosh et al. Textbook of Pediatries, 7th edn. 2008.

Biopsy result (day 32): cholestasis with cholangiolitis and interlobular bile

duct destruction. WBC, White Blood Cell; RBC, Red Blood Cell; Hb, hemo-

globin; Ht, hematocrit; Reti, Reticulocytes; Plt, Platelets; PT-INR, Pro-

thrombin Time-International Normalized Ratio; APTT, activated partial

thromboplastin time; TP, Total Protein; Alb, Albumin; AST, Aspartate tran-

saminase; ALT, Alanine aminotransferase; GGT, Gamma-glutamyl

transpeptidase; TBA, Total Bile Acids; ALP, Alkaline phosphatase; LAP,

Leucine aminopeptidase; LDH, Lactate dehydrogenase; CK, Creatinine

Kinase; TSB, Total Serum Bilirubin; CB, Conjugated Bilirubin; CRP,

C-reactive protein.

R Taba et al. Cholestasis presenting as cholangiolitis

Journal of Paediatrics and Child Health 48 (2012) E126–E131© 2010 The AuthorsJournal of Paediatrics and Child Health © 2010 Paediatrics and Child Health Division (Royal Australasian College of Physicians)

E127

the cholestasis. Perhaps because of steroid treatment, hislength remained below the first percentile for eight months. Afollow-up ABR (performed at 12 months) and brain MRI (at18 months) were normal. At present (age, 24 months), hislength and psychomotor development are normal.

Discussion

Significant early-onset cholestasis occasionally accompaniescases of HDN.1,5 Several case reports describe cholestasis as acomplication of HDN.1–3,5 In our case, histological changes wereaccompanied by many CD8+ T cells in the portal area and inter-lobular bile ducts (Fig. 1).

First, although we suspected that the two ETs might havecaused transfusion-associated graft-versus-host disease (GVHD),the transfused packed red blood cells and platelets had beenfiltered to reduce leukocytes and were routinely irradiated

(50 Gy). Moreover, cholangiolitis could be a complication of theprocedure if the ETs were to be performed via the umbilicalvenous catheters. However, both ETs were performed via thebasilic vein in the arm.

Second, we hypothesised that maternal microchimeric cellsmight act as the stimulus of host cell proliferation in a GVHD-like process.6 As maternal cells are commonly found in foetaltissues and maternal microchimerism may result in foetalimmunologic tolerance,7 the foetal immunological environmentshould be examined with care. Although double fluorescencein situ hybridisation detected XX hybridisation signals in seve-ral cells, liver biopsy specimens from controls were notavailable. Quantitative comparison of maternal cells withcontrol cells is indispensable for determining whether such cellsare alloresponsive.

UDCA and PB have been used successfully for treatment ofcholestasis in HDN cases.1,4,5 Although not tested in randomisedcontrolled trials, steroids have long been used as adjunct treat-ment in postoperative biliary atresia6 and HDN-related cholesta-sis.2 Although use of steroids in HDN-related cholestasis hasbeen advocated,2 their class and dosage are not usuallyreported1,3–5 because the cholestasis is mild. Possibly such casesof steroids used to reduce conjugated hyperbilirubinemia mayalso involve cholangiolitis as the aetiology of refractorycholestasis as was the case in our patient. There was no basis tostart steroids except for the biopsy results in our case. If we hadnot implemented the liver biopsy, we would not have had theoption of commencing steroids. We used the class and dose ofsteroid reported for the treatment of neonatal autoimmunehaemolytic anaemia.8

Severe Rh isoimmunisation is rare in Japan, owing to theavailability of preventive options, including intra-uterine trans-fusion before 34 weeks of gestation and induction of labour

Fig. 1 (a) Many lymphocytes infiltrate the portal area, causing interlobular

bile duct destruction (arrowheads) as manifested by bile plugs, degenera-

tion of hepatic cells and swelling of hepatic cells (haematoxylin–eosin stain;

magnification’10, ‘200 (inset)). There was no bile ductular proliferation. (b)

Immunohistochemical analysis in the portal area of the infant’s liver. A

number of CD8+ T cells (arrows) are found at the periphery of the interlobu-

lar bile ducts (CD8 immunohistochemical staining; magnification’10).

Fig. 2 Hepatobiliary scintigraphy (a) before prednisolone therapy (day 35)

demonstrating no tracer was transferred to the intestines until 30 h after

administering the tracer and (b) four days after initiation of methylpredniso-

lone therapy (day 42), demonstrating that the tracer was eliminated no later

than 60 min after tracer administration.

Cholestasis presenting as cholangiolitis R Taba et al.



E128

before 38 weeks of gestation. Only a small number of pregnantwomen in Japan refuse prenatal care and thus fail to receiveperinatal management of Rh isoimmunisation.

In conjugated hyperbilirubinemia, phototherapy increasesthe risk of bronze baby syndrome. Since the latter is generallybenign, the former should not be a contraindication for photo-therapy.4 Cholestasis can reduce the effectiveness of photo-therapy since its products are excreted in bile.4 In theory, directbilirubin does not cross the blood–brain barrier.3,5 However,the contribution of CB to kernicterus is not clear.3–5 Neverthe-less, when CB is significantly elevated, there is no bilirubinthreshold at which intervention is recommended.3 The Ameri-can Academy of Pediatrics guidelines recommend decisions forphototherapy, and ET should be based on the total level ofbilirubin.9 We based our decisions for ET on the total level ofbilirubin.

The use of 0.5–1.0 g/kg IVIg in Rh-HDN should be consideredif the TSB level is rising despite intensive phototherapy or if TSBlevel is within 34–51 mmol/L of the exchange level.9

Once cholestasis established, further investigations should beperformed in a stepwise manner to establish the specific cause ofcholestasis (Fig. 4).4 Most important for clinicians is to distin-guish extrahepatic cholestasis from intrahepatic causes. Liverbiopsy is the single most definitive investigation in the evalua-tion of neonatal cholestasis.4 Medical management of cholesta-sis is mostly supportive and does not alter the natural course.4,5

Although cholestasis due to HDN has been reported to resolvewithin a month, Allgood reported two cases of cholestasis due toHDN that persisted for over three months.1 Our case could alsohave taken much longer to resolve than the reported cases until

resolution if we had not adopted the steroid therapy in our case.Severe haemolysis and conjugated hyperbilirubinemia havebeen noted to be predisposing factors to cholelithiasis in infancy,leading to biliary sludging and/or development of gallstones.5

Repeated ultrasounds to monitor cholelithiasis are required inaddition to laboratory monitoring. Liver biopsy is indispensablefor a full-term infant with intrahepatic cholestasis to demon-strate the definitive diagnosis in the evaluation of neonatalcholestasis4 despite the fact that we have nothing to do but waituntil the conjugated hyperbilirubinemia is resolved regardless ofthe result.

In the present case, cholangiolitis, in addition to bile inspis-sation and/or hepatocellular damage, led to persistent cholesta-sis. In such cases, corticosteroids might be adjunctive treatmentfor refractory conjugated hyperbilirubinemia.

Summary

This case of refractory intrahepatic cholestasis was in a newbornpresenting with cholangiolitis disclosed by immunohistochemi-cal investigation showing a number of CD8+ lymphocytes withinthe portal tract, damage to the interlobular bile duct and anti-Eisoimmunisation.

Bilirubin levels decreased dramatically after 14 days of corti-costeroid therapy (methylprednisolone, 2 mg/kg/day), implicat-ing an inflammatory process (e.g. cholangiolitis) as the cause ofrefractory cholestasis.

Corticosteroids might be adjunctive treatment for refractoryconjugated hyperbilirubinemia in HDN.

0 10 20 24 30 32 34 45 70 4 11 22 36 49 64 78 106 125

Hours Days

Phototherapy

IVIG 1 g/kg

ET 160 ml/kg/dose

UDCA 20 mg/kg/D and PB 5 mg/kg/D

Methylprednisolone (0.25-2 mg/kg/D)

*

0

100

200

300

400

500

600

TSB CB

* Operative cholangiographyand liver biopsy

Tot

al b

iliru

bin

and

co

nju

gate

d b

iliru

bin

leve

l, µ

mol

/L

Fig. 3 Bilirubin level associated with interventions.



E129

References

1 Allgood C, Bolisetty S. Severe conjugated hyperbilirubinaemia andneonatal haemolysis. Int. J. Clin. Pract. 2006; 60: 1513–4.

2 Dunn PM. Obstructive jaundice and hemolytic disease of the newborn.Arch. Dis. Child. 1963; 38: 54–61.

3 Smits-Wintjens VE, Walther FJ, Lopriore E. Rhesus haemolytic diseaseof the newborn: postnatal management, associated morbidity andlong-term outcome. Semin. Fetal Neonatal Med. 2008; 13:265–71.

4 Venigalla S, Gourley GR. Neonatal cholestasis. Semin. Perinatol. 2004;28: 348–55.

Fig. 4 Flow diagram of conjugated hyperbilirubinemia in full term infant and interventions4 (midufied).

Cholestasis presenting as cholangiolitis R Taba et al.



E130

5 McAdams RM, Dotzler SA, Winter LW, Kerecman JD. Severehemolytic disease of the newborn from anti-e. J. Perinatol. 2008; 28:230–2.

6 Muraji T, Hosaka N, Irie N et al. Maternal microchimerism in underlyingpathogenesis of biliary atresia: quantification and phenotypes ofmaternal cells in the liver. Pediatrics 2008; 121: 517–21.

7 Mold JE, Michaëlsson J, Burt TD et al. Maternal alloantigens promotethe development of tolerogenic fetal regulatory T cells in utero.Science 2008; 322: 1562–5.

8 Motta M, Cavazza A, Migliori C, Chirico G. Autoimmune haemolyticanaemia in a newborn infant. Arch. Dis. Child. Fetal Neonatal Ed. 2003;88: F341–2.

9 American Academy of Pediatrics Subcommittee on Hyperbilirubinemia.Management of hyperbilirubinemia in the newborn infant 35 or moreweeks of gestation. Pediatrics 2004; 114: 297–316.



E131

refractory cholestasis presenting as cholangiolitis in an rh (e)-incompatible neonate

Documents