Immunostaining in PFIC – how does it help in diagnosis?
By Alex Knisely (go to foot of section for affiliation, contact details, and for pictures of immunostained
liver biopsy materials)
whose child has been diagnosed with PFIC, “progressive familial intrahepatic cholestasis”, immediately wants to
know: What does that mean?
does PFIC stand for?”, but “What does this diagnosis mean for my baby, and for our family?”
let me be clear: When I write “PFIC”, I mean a disorder in which,
despite jaundice [“conjugated hyperbilirubinemia”], serum concentrations of gamma-glutamyl transpeptidase [GGT]
activity do not rise. “Low-GGT PFIC” is a very different kettle of
fish from “high-GGT PFIC. Check with your child’s doctors if you
are not clear on this point, it is worth the telephone call.)
different kinds of PFIC are well-defined. That is, the gene responsible is known
for each of them, even if all the interactions and functions of the protein encoded by that gene are not yet sorted out. One of these is a disorder limited to the liver.
One of these is a disorder that affects many body systems.
in itself is a good reason to know which sort of PFIC your child has; down the road, most children with PFIC will come to
liver transplantation. Will the transplant “cure” your child’s
disorder? (By “cure”, I mean:
“Replace one disease with another one that is much easier to deal with”, not “return-to-absolute-and-total-health”.) Or will the transplant unmask a new set of problems in organs other than the liver?
one example of why exact diagnosis, that is, disease identification, can be important: It helps give a more specific prognosis, that is, forecast of what your child and you can
immunostaining be used to help your child’s doctors toward an exact diagnosis?
uses antibodies to mark a specific antigen, in this case a protein, within cells or tissues.
Immunostaining for BSEP is one way to try to distinguish between the two kinds of PFIC.
In PFIC, type 1 (PFIC-1), bile salt export pump is normally
expressed within the liver – at least as far as immunostaining can discern! But
in PFIC, type 2 (PFIC-2), BSEP is not detectable by immunostaining (in almost every instance that our team, and other groups
of researchers, have studied to date).
is particularly helpful because PFIC-1 and PFIC-2 can look remarkably like each other, both in how the sick child behaves
and under the microscope. Indeed, PFIC-1 and PFIC-2 were separated and distinguished
only within the last ten years.
A bit of
information about BSEP and its role in bile formation: BSEP moves bile salts
from inside the liver cell, or hepatocyte, into the tiniest twigs of the “biliary tree”. These are called “bile canaliculi”. They lie among
hepatocytes, forming a sort of network. Their contents, which are the earliest
form of bile, drain into bile ducts and, through bile ducts, into the small bowel. If
BSEP does not work, or is absent, bile salts accumulate within hepatocytes and cause damage.
This damage then leads to failure of hepatocytes to carry out other normal processes – such as moving bile pigment
(very different from bile salts!) into bile. Body-wide, jaundice (back-up into
the blood plasma of bilirubin, that is, bile pigment) and itching (back-up into the blood plasma of bile salts) result. Within the liver, inflammation, cell death, and scarring result.
canaliculus is lined, and defined, by a particular region of the cell membrane of the hepatocyte. BSEP passes back and forth through that region much as a shoelace passes through the eyelets of your shoe,
from one instep to the other, back and forth across the tongue. If the lace is
not fed through the eyelets properly (if the gene encoding BSEP is mutated in a way that leads to abnormal handling of BSEP
within the hepatocyte), the shoe fits poorly (“hepatocellular injury”).
Immunostaining is a very specific tool that lets a microscopist see how well, if at all, the hepatocyte’s shoelace
is threaded and tied.
of synthesizing, and transporting, and folding, and inserting BSEP into the canalicular membrane is monstrously complicated. Our team believes that mutation in ABCB11, the gene encoding BSEP, is thus very likely to lead to absence of BSEP at the canaliculus – just because so
many things can go wrong. With ABCB11 mutation, if you look at
the tongue of the shoe (the canaliculus) to find the criss-cross of the lace (BSEP) above it, as a rule you find nothing;
the lace never makes it to the insteps.
(BSEP is expressed only in the liver. PFIC-2 is
thus a liver-limited disease, and liver transplantation in PFIC-2 can provide a “cure” – defined as above. PFIC-1, however, is a multi-system disorder.)
What if immunohistochemical study finds no BSEP in a liver biopsy specimen from a child
with PFIC, although other, similar proteins are detected in a normal pattern? This
means that PFIC-2 is more likely than PFIC-1 in the child from whom the specimen came.
In turn, ABCB11 is the gene likely to be abnormal in that child. Immunostaining can
tell doctors: “Check here first!”
Without demonstrable BSEP, ABCB11 is the gene toward which that child’s doctors should direct mutational analysis
if a family wants absolute security in diagnosis. Mutational analysis also is
of value if antenatal diagnosis is an option for other children in that family: To
look for mutations known to be present in an affected child can give information on whether or not an unborn child will have
the same disorder.
Finally, to demonstrate that BSEP is normally expressed in a liver biopsy specimen from
a child with PFIC generally means that PFIC-1 is more likely in that child than is PFIC-2.
As with many things in medicine, this interpretation is not absolutely certain.
Absolute certainty requires mutational analysis of genes. But to see normal
BSEP expression will point mutational analysis toward ATP8B1, the gene mutated in PFIC-1. As
with PFIC-2, to know the results of immunostaining can save much effort and time, and can get a family the hoped-for answer
Alex Knisely, MD
Institute of Liver Studies
King’s College Hospital
London SE5 9RS
firstname.lastname@example.org e-mail for comments or questions, please
(all images photographed at the same magnification, click to enlarge):
“Control liver” showing normal canaliculi expressing BSEP. Note the
brown, regular network throughout the tissue section.
liver” showing disordered and damaged hepatocytes; no network of canaliculi is outlined.
liver” showing disordered and damaged hepatocytes with a damaged and irregular canalicular network. This immunostain is for MRP2, a protein with a structure similar to that of BSEP. Liver injury has altered the appearance of the canalicular network, but to see MRP2 along canaliculi means
that a general defect in handling proteins like BSEP and MRP2 is not present.
This means, in turn, that the absence of BSEP is a specific phenomenon, pointing toward mutation in ABCB11.