The usefulness of bone marrow aspiration in the
diagnosis of Niemann–Pick disease type C in
infantile liver disease
A F Rodrigues, R G Gray, M A Preece, R Brown, F G Hill, U Baumann, P J McKiernan
............................................................... ............................................................... .
See end of article for
Dr A F Rodrigues, Liver
Birmingham, UK; kvtsi@
Accepted 23 May 2006
Published Online First
31 May 2006
Arch Dis Child 2006;91:841–844. doi: 10.1136/adc.2005.088013
Background: Niemann–Pick disease type C (NPC) is a fatal, autosomal recessive lysosomal storage
disease which may present in infancy with cholestatic jaundice and/or hepatosplenomegaly. In cholestatic
patients with splenomegaly, a bone marrow aspirate has been advocated as a relatively accessible tissue
to demonstrate storage phenomena. Typically in patients with NPC, macrophages with abnormal
cholesterol storage, so called foam cells, can be detected in the bone marrow.
Aim: To review our experience of bone marrow aspiration in children with NPC presenting with infantile
Methods: A retrospective analysis of 11 consecutive children (8 males) from Birmingham Children’s
Hospital with NPC presenting with infantile liver disease was undertaken. The diagnosis of NPC was
confirmed in all cases by demonstrating undetectable or low rates of cholesterol esterification and positive
filipin staining for free cholesterol in cultured fibroblasts.
Results: The median age at presentation was 1.5 months (range 0.5–10). Bone marrow aspirates showed
storage cells in only 7/11 cases. Bone marrow aspirates which had storage cells were undertaken at a
median age of 11 months while those with no storage cells were undertaken at median age 2.3 months.
The overall sensitivity of bone marrow aspirates for detecting storage cells in children presenting with
infantile liver disease was 64%; however, for children who had bone marrow aspirates in the first year of
life it was only 57%.
Conclusions: The sensitivity of bone marrow aspirate for the diagnosis of NPC disease in patients
presenting with infantile liver disease was lower than previously reported. Where NPC is suspected
clinically, definitive investigations should be undertaken promptly. There is a need to develop sensitive
screening methods for NPC in children presenting with infantile liver disease.
hepatosplenomegaly, liver dysfunction, progressive neurode-
generation, and a vertical supraopthalmoplegia.1These occur
secondary to lysosomal accumulation of unesterified choles-
terol; the disease is caused by mutations in one of two
underlying genes, NPC1 or NPC2.2 3
Between 45% and 65% of NPC cases present with neonatal
liver disease4–6manifest as conjugated hyperbilirubinaemia
and/or hepatosplenomegaly. Cholestasis resolves sponta-
neously by 2–4 months in the majority of infants, but in
about 10% of cases it may lead to rapidly progressive liver
Diagnosing NPC is difficult in these infants as the
differential diagnosis is wide and neurological features will
not have developed. NPC may mimic conditions such as
biliary atresia and it is important to have an accurate, quick,
and reliable test to help reach a diagnosis. This is important
to prevent unnecessary investigations as well as to guide
prognosis, genetic counselling, and antenatal diagnosis.8
Furthermore, in those infants who develop liver failure, an
early diagnosis may help avoid unnecessary liver transplan-
The definitive method for diagnosing NPC is the demon-
stration of undetectable or low rates of cholesterol esterifica-
tion accompanied by excess storage of free cholesterol by
filipin staining in cultured fibroblasts11–13or by the detection
of two pathogenic mutations.2 3These tests are expensive,
available only at a few specialised centres and may take a
iemann–Pick disease type C (NPC) is a fatal, auto-
somal recessive, lysosomal storage disorder charac-
terisedbya variablephenotypethat includes
long time for results to become available. This makes them
impractical as first line/screening investigations. Early diag-
nosis rests on maintaining a high level of clinical suspicion
and demonstrating characteristic storage cells, usually in liver
and/or bone marrow tissue.
Liver histology alone is insufficient for this purpose, as
histological features diagnostic of NPC are found on liver
biopsy in only 50% of cases, even when the diagnosis is
already known.5Bone marrow aspiration has been shown
retrospectively to be a sensitive and relatively non-invasive
method for demonstrating storage material in children with
confirmed NPC.5As a result, bone marrow aspiration has
been recommended in the investigation of unexplained
neonatal cholestasis, especially if accompanied by persistent
splenomegaly.5 6There is however little information on the
diagnostic yield of bone marrow aspiration when it is
undertaken at the time of presentation with neonatal liver
The aim of this study was to review our experience of bone
marrow aspirates in infants with NPC presenting with
infantile liver disease.
SUBJECTS AND METHODS
A retrospective review of children proven to have NPC who
presented with infantile liver disease between 1985 and 2004
was undertaken. During this time approximately 750 cases of
infantile liver disease were assessed at the Liver Unit at
Birmingham Children’s Hospital. Children were investigated
using a structured protocol which evolved throughout the
time of this study.7In addition to excluding recognised
surgical, metabolic, and infective causes of neonatal liver
disease, percutaneous liver biopsy was undertaken in the
great majority of cases. Bone marrow aspiration was a
discretionary second line investigation indicated where there
was unexplained splenomegaly or where no cause of
cholestasis had been discovered after first line investigations.
All children investigated for infantile liver disease were
followed up clinically until a specific diagnosis was estab-
lished or until they had made a complete clinical and
The diagnosis of NPC was established in all cases by
demonstrating undetectable or low rates of cholesterol
esterification accompanied by demonstrating excess storage
of free cholesterol by filipin staining in cultured fibroblasts
using published methods.13
Data pertaining to their clinical and biochemical status at
the time of initial presentation with infantile liver disease,
bone marrow biopsy reports with their timings, and the age
at which the diagnosis of NPC was confirmed were obtained.
A haematologist re-examined all the bone marrow aspirate
samples, without knowledge of the original report. The liver
biopsies were also re-examined by a histopathologist and
these current reports were used for the analysis.
Liver biopsy specimens were fixed in formalin and
processed into paraffin wax for routine sections, stained
with haematoxylin and eosin, and routine special stains
including PAS/PASD. A liver biopsy was regarded as
diagnostic for Niemann–Pick disease where foamy macro-
phages were visible on light microscopy highlighted in relief
on PAS staining. Liver biopsy specimens were also preserved
in glutaraldehyde for electron microscopy. Bone marrow
aspirates were stained with Wright’s stain and examined by
Results are presented in table 1. Eleven children (eight male)
were identified, accounting for approximately 1.5% of cases
of infantile liver disease seen at our centre. The median age at
initial presentation was 1.5 months (range 0.5–10). Seven
children presented with conjugated hyperbilirubinaemia and
hepatosplenomegaly and four had only hepatosplenomegaly.
Their median bilirubin level at presentation was 125 mmol/l
(range 5–192). The median age at eventual diagnosis of NPC
was 10 months (range 1.5–47).
Bone marrow aspirates were done at a median age of
10 months (range 1.5–47). In seven cases the bone marrow
aspirate was undertaken at the time of initial evaluation of
infantile liver disease. In the remainder this was carried out
as a delayed second line investigation where clinical concern
Seven children (median age at biopsy 11 months; range
1.5–47) had characteristic storage cells found, while four
children (median age at biopsy 2.3 months; range 1.5–16)
had normal bone marrow aspirates. The bone marrow
aspirates were re-examined for the purpose of this study.
All samples were found to be of diagnostic quality with
adequate cellularity. The original findings were confirmed in
all cases with no ambiguous cases discovered.
The overall sensitivity of bone marrow aspirates for
detecting storage cells in children presenting with infantile
liver disease was 7/11 (64%); however for children who had
their bone marrow aspirates in the first year of life it was only
Fourteen liver biopsies were carried out on 10 children.
One child presenting at 9 months with hepatosplenomegaly
had a diagnostic bone marrow aspirate and a liver biopsy was
not undertaken. Nine children had liver biopsies done at the
time of their initial presentation at a median age of
1.5 months (range 1.5–11). In only one child was this
diagnostic of Niemann–Pick disease (at 11 months of age).
The other eight biopsies showed a non-specific pattern typical
of idiopathic neonatal hepatitis, with in addition evidence of
bile duct paucity in four. In seven cases electron microscopy
was undertaken, of which five had abnormal findings. These
consisted of abundant myelin figures, which were suggestive
but not diagnostic for Niemann–Pick disease. One child who
initially presented at 2 months of age did not have invasive
investigations in infancy. He subsequently underwent liver
biopsy at 29 months because of persistent hepatosplenome-
galy. This showed classical diagnostic features of Niemann–
Four children had repeat liver biopsies at a median age of
16.5 months (range 5.5–24) because of persisting unex-
plained splenomegaly. Three of these now showed diagnostic
features of Niemann–Pick disease; one biopsy was of
insufficient quality to allow meaningful interpretation. The
sensitivity of liver histology for detecting diagnostic features
of NPC in children presenting with infantile liver disease was
5/14 (36%); however where the liver biopsies were under-
taken in the first year of life it was only 2/10 (20%).
In our centre, bone marrow aspirates have proved to be a
poor screening test for NPC presenting with infantile liver
disease with a sensitivity of only 57% for those done in the
first year of life. Strong clinical suspicion remained crucial in
establishing the diagnosis and initiating definitive investiga-
Previous studies and correspondence have suggested that
false negative bone marrow aspirates for NPC are unusual.5 14
Timing and results of biopsies in infants presenting with infantile liver disease
Age at bone
Age at initial
Liver biopsy result
Age at repeat
Liver biopsy result
LMLM EM PBD
Details of patients listed according to their age at the time of bone marrow aspirate.
+, presence of storage cells; 2, no storage cells seen; s, suggestive; n, non-suggestive; x, not done.
LM, light microscopy; EM, electron microscopy; PBD, paucity of bile ducts.
842 Rodrigues, Gray, Preece, et al
This is at variance with our experience. Sampling or
interpretive errors appear unlikely given that the bone
marrow aspirate slides were reviewed by an experienced
haematologist, who confirmed the diagnostic quality, ade-
quacy of cellularity, and the original findings of all the
A major strength of the current study is that a clearly
defined population was assessed prospectively using a
structured protocol with a high rate of clinical follow up.
Therefore the chance of further missed cases was low. In
contrast, previous studies have been retrospective, based on
children in whom the diagnosis of NPC disease was already
established, often on the basis of demonstrating storage
material.5We have found that storage cells are more likely to
be found if the bone marrow aspirate was performed later.
So, while a positive bone marrow aspirate is specific for NPC,
marrow storage cells in NPC may only become apparent as
the disease evolves.15If this is so, relying solely on early bone
marrow aspiration for diagnosis of NPC may be intrinsically
flawed. Unfortunately our findings cannot suggest an
obvious cut-off age above which bone marrow aspirate is
more likely to be diagnostic.
The overall sensitivity of liver biopsies for detecting storage
cells in these children was 36%, which is comparable to
earlier studies.5Our findings suggest that liver histology
findings in NPC also develop with time. The earliest liver
biopsy that revealed the typical light microscopic features of
Niemann–Pick disease was obtained at 5.5 months. Thus,
early biopsies may also miss the diagnosis. This is a similar to
what we have observed with bone marrow aspirates, and is
consistent with previous reports of liver histology in NPC.16
Electron microscopy was suggestive of Niemann–Pick
disease even before characteristic changes were seen in the
corresponding light microscopy results. Although myelin
figures can be a non-specific finding in neonatal hepatitis, if
they are present in large numbers and not confused with
whorled structures seen in bile whenever there is cholestasis,
they can be a helpful marker for targeting further investiga-
Interestingly, 4/5 patients biopsied before 2 months of age
showed paucity of bile ducts; this resolved in two children
who had follow up biopsies. Non-syndromic bile duct paucity
is a non-specific finding and can occur from a variety of
infective, metabolic, and genetic causes.17–20This has only
been reported in one previous case of NPC and may be more
common than previously suspected. The mechanism of bile
duct paucity in NPC is unclear, but may be related to the
production of abnormal bile acids which have been reported
in NPC.21Whether these develop due to unusual metabolism
of cholesterol as a consequence of its abnormal subcellular
distribution or as a reflection of involvement of NPC1 in the
conversion of cholesterol to bile acids is unknown. Our
retrospective study cannot shed any light on this matter but it
does emphasise the importance of considering NPC in the
differential diagnosis of bile duct hypoplasia associated with
raised c-glutamyl transferase.
Our findings highlight the need for a reliable screening test
for NPC in children with infantile liver disease to determine
who will require definitive investigations. Unfortunately
there is no proven method. Serum or plasma chitotriosidase,
an endo-b-glucosaminidase secreted by activated macro-
phages, has been shown to be moderately elevated22 23in
proven cases of NPC. This test is relatively easy and may be
useful as a screening tool, although it has not yet been
evaluated for this purpose.
In conclusion, bone marrow aspirates are not reliable as a
screening test in the diagnosis of NPC presenting with
infantile liver disease. While a positive result is a useful
pointer to NPC, a negative result cannot exclude the
diagnosis. In children with a high index of clinical suspicion,
i.e. especially those presenting with cholestasis and unex-
plained splenomegaly, skin biopsy for fibroblast culture
should be undertaken without delay to enable definitive
tests to be performed. Reliable screening tests for NPC are
A F Rodrigues, U Baumann, P J McKiernan, Liver Unit, Birmingham
Children’s Hospital, Birmingham, UK
R G Gray, M A Preece, Clinical Chemistry, Birmingham Children’s
Hospital, Birmingham, UK
R Brown, Histopathology Department, Birmingham Children’s Hospital,
F G Hill, Haematology Department, Birmingham Children’s Hospital,
Competing interests: none declared
1 Fink JK, Filling-Katz MR, Sokol J, et al. Clinical spectrum of Niemann-Pick
disease type C. Neurology 1989;39:1040–9.
2 Carstea ED, Polymeropoulos MH, Parker CC, et al. Linkage of Niemann-Pick
disease type C to human chromosome 18. Proc Natl Acad Sci U S A
3 Vanier MT, Duthel S, Rodriguez-Lafrasse C, et al. Genetic heterogeneity in
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4 Vanier MT, Wenger DA, Comly ME, et al. Niemann-Pick disease group C:
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5 Kelly DA, Portmann B, Mowat AP, et al. Niemann-Pick disease type C:
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9 Gartner JC Jr, Bergman I, Malatack JJ, et al. Progression of neurovisceral
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What is already known on this topic
N Diagnosing NPC in infants presenting with liver disease
can be difficult and a high level of clinical suspicion is
N The definitive diagnostic method is to demonstrate
abnormal cholesterol esterification in cultured fibro-
blasts or to detect pathogenic mutations
What this study adds
N Bone marrow aspirates are not reliable as a screening
test for NPC presenting with infantile liver disease
N In children with a high index of clinical suspicion,
definitive tests (skin biopsy for fibroblasts culture)
should be undertaken without delay
Bone marrow aspiration in Niemann-Pick disease843
14 Patterson MC, Pentchev PG. Diagnosis of Niemann-Pick disease type C.
J Pediatr 1994;124:655–6.
15 Yan-Go FL, Yanagihara T, Pierre RV, et al. A progressive neurologic disorder
with supranuclear vertical gaze paresis and distinctive bone marrow cells.
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neonatal cholestasis at a North American Center. J Pediatr Gastroenterol Nutr
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bile ducts: light and electron microscopic evaluation of sequential liver
biopsies in early childhood. Hepatology 1986;6:890–901.
18 Kocak N, Gurakan F, Yuce A, et al. Nonsyndromic paucity of interlobular bile
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19 De Tommaso AM, Kawasaki AS, Hessel G. Paucity of intrahepatic bile
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20 Yehezkely-Schildkraut V, Munichor M, Mandel H, et al. Nonsyndromic
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23 Guo Y, He W, Boer AM, et al. Elevated plasma chitotriosidase activity
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IMAGES IN PAEDIATRICS............................................................... ...............
Extreme subcutaneous emphysema of eyelids
evening, parents had noticed slight
swelling over the neck and right eye,
which during the night worsened and
spread to the left eye and chest. There
was no history of fever, cough, breath-
ing difficulty, trauma, or surgery; how-
ever, she had been constipated for the
last four days. On examination she was
in respiratory distress with gross swel-
ling of the face and eyelids (fig 1). Her
temperature was 100˚F, pulse 130, and
respiratory rate 65 per minute. Crackles
were palpable over the neck and chest; x
ray examination showed pneumome-
diastinum, subcutaneous emphysema
of chest, neck, and orbits (fig 2). She
was treated conservatively; the swelling
subsided considerably during the next
10 days and disappeared completely
after six weeks (fig 3).
Orbital emphysema is an uncommon
complication of facial injuries or opera-
tive procedures.1 2It can also be caused
by straining such as the Valsalva man-
oeuvre or even blowing the nose.3Since
there was a history of constipation in
our patient, it was assumed that strain-
ing led to interstitial and subsequently
orbital emphysema. Emphysema usually
resolves spontaneously in 2–3 weeks,
therefore no active treatment is needed,
even when it is severe.4
e describe a previously healthy
14 month old girl with a one
day history of bilateral perior-
M Azam, M Jamal, N Bhatti
Children’s Hospital, Pakistan Institute of
Medical sciences, Islamabad, Pakistan;
Competing interests: none
Parental consent was obtained for publication
of the figures
1 Ortak T, Mithat Akan I, Sensoz O. Subcutaneous
emphysema of the eyelid and orbital cone after
maxillary fracture. Ann Plast Surg 2001;46:195.
2 Hasel R, Arora SK, Hickey DR. Intraoperative
complications of laparoscopic cholecystectomy.
Can J Anaesth 1993;40:459–64.
3 Mohan B, Singh KP. Bilateral subcutaneous
emphysema of the orbits following nose blowing.
J Laryngol Otol 2001;115:319–20.
4 Zachariades N, Mezitis M. Emphysema and
similar situations in and around the maxillo-facial
region. Rev Stomatol Chir Maxillofas
the eyelids; the eyes were shut tightly, and the
The patient had extreme swelling of
emphysema of the orbits.
Skull x ray showing subcutaneous
swelling of eyelids and face.
Normal face of the child without
844 Rodrigues, Gray, Preece, et al