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)

Everyone whose child has been diagnosed with PFIC, “progressive familial intrahepatic cholestasis”, immediately wants to know: What does that mean?

Not “What does PFIC stand for?”, but “What does this diagnosis mean for my baby, and for our family?”

(Please 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.)

Two 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.

That 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?

This is 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 expect.

How can immunostaining be used to help your child’s doctors toward an exact diagnosis?

Immunostaining 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).

This observation 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.

The 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.

The process 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 faster.

Alex Knisely, MD

Consultant Histopathologist
Institute of Liver Studies
King’s College Hospital
Denmark Hill
London SE5 9RS United Kingdom e-mail for comments or questions, please