Laboratory

Diagnosis of
Hepato-biliary
Diseases
ERIC M. MIRANDILLA MD DPSP
 Discuss the principles behind laboratory tests
Discuss for liver function.

Objectives Discuss the advantages as well as limitations of

Discuss each test.

Correlate the different hepatobiliary diseases

Correlate with specific patterns of abnormalities seen the
laboratory parameters.
Evaluation of Liver Function
THE LIVER
The liver is the largest organ of the body, weighing 1–1.5 kg and
representing 1.5–2.5% of the lean body mass.

Majority of the cells are hepatocytes.

Using light microscopy, the liver appears to be organized in lobules, with

portal areas at the periphery and central veins in the center of each
lobule.
THE LIVER
Zone 1 – hepatic arterial and portal venous blood entering
the acinus from the portal areas

Zone 2 – the intervening hepatocytes

Zone 3 – through the sinusoids to the terminal hepatic veins

NORMAL LIVER FUNCTION

Hepatocytic system – metabolic functions

Hepatobiliary system – metabolism of bilirubin

Reticuloendothelial system – involved with the immune system

NORMAL BILIRUBIN METABOLISM

Bilirubin is the major metabolite of heme.

250-350mg of bilirubin is produced daily in healthy adults and 85% are

derived from turnover of senescent red blood cells

Biliverdin is reduced to bilirubin by biliverdin reductase that is a NADPH-

dependent enzyme.
NORMAL BILIRUBIN METABOLISM

Bilirubin is transported in the portal system and is bound mainly to

albumin.

Clearance of unconjugated bilirubin at normal values is about

5mg/kg/day

Bilirubin is commonly in its trans- form and is highly insoluble to water

 NORMAL BILIRUBIN METABOLISM

Light can cause photoisomerization of biirubin, transforming it into its

cis- form, making it more soluble to water and easily excreted in the
urine.

There are two mechanisms by which bilirubin enters the hepatocytes:

◦ Passive diffusion
◦ Receptor – mediated endocytosis
NORMAL BILIRUBIN METABOLISM
Bilirubin is further metabolized by intestinal bacteria and forms the
compound urobilinogen and urobilin which can be reabsorbed in the
gut.
NORMAL BILIRUBIN METABOLISM
 CAUSES OF ELEVATED SERUM
LEVELS OF UNCONJUGATED
BILIRUBIN

Hemolysis

Gilbert’s syndrome and the Crigler-Najjar Syndrome

◦ Gilbert’s syndrome - characterized by mild unconjugated bilirubinemia.
◦ Total bilirubin is unconjugated and is typically elevated to 2–3 mg/dL; levels can increase further
with fasting but seldom exceed 5 mg/dL.
 CAUSES OF ELEVATED SERUM
LEVELS OF UNCONJUGATED
BILIRUBIN

◦ Crigler-Najjar Syndrome - characterized by high serum levels of unconjugated

bilirubin

◦ Type I - the unconjugated hyperbilirubinemia exceeds 5-20mg/dL causing jaundice.

◦ Type II - enzyme activity is approximately 10% of normal.

 CAUSES OF ELEVATED SERUM
LEVELS OF CONJUGATED BILIRUBIN

Excretion deficits: Dubin-Johnson Syndrome

◦ Associated with increased plasma conjugated bilirubin, typically with mild

jaundice (total bilirubin 2–5 mg/dL), and intense dark pigmentation of the
liver due to accumulation of lipofuscin pigment.
CAUSES OF ELEVATED SERUM LEVELS OF
CONJUGATED BILIRUBIN
Biliary obstruction
◦ Cholelithiasis - concomitant rise in alkaline phosphatase
◦ Inflammatory conditions of the biliary tract (ascending cholangitis) - rise in
direct bilirubin often exceeds 5 mg/dL. Elevation of alkaline phosphatase to
levels of 200-300 IU/L.
◦ Hepatitis - elevation of both direct and indirect bilirubin.
LABORATORY TEST FOR
BILIRUBIN
Bilirubin is measured using diazotized sulfanilic acid

Caffeine or methanol are used to speed up the reaction of

unconjugated bilirubin.

Direct bilirubin should be 0-0.1 mg/dL in normal individuals, with rare

values of 0.2 mg/dL in the absence of liver or biliary tract disease.
OTHER METABOLIC
TESTS
Ammonia

◦ The liver is the only organ that contains the critical enzymes for the Krebs–
Henseleit or urea cycle, in which ammonia, a toxic substance, is ultimately
converted into urea.
◦ ornithine carbamoyltransferase (OCT).
OTHER
METABOL
IC TESTS
OTHER METABOLIC
TESTS
Assays for Ammonia
Glutamate dehydrogenase (enzymatic assay)

Alkaline pH buffers (dry slide method)

Arterial blood is the preferred specimen for measurement of ammonia.

OTHER METABOLIC
TESTS
Lipids

◦ Cholesterol and other Lipids - decrease in the HDL, particularly the HDL 3 (but
often not the HDL2)
◦ In alcohol liver injury, HDL, especially HDL3, may be elevated if alcohol ingestion continues.
◦ In cholestasis, cholesterol levels in serum can become markedly elevated.
OTHER METABOLIC
TESTS
◦ Bile Salts

◦ Bile salts are important because they contain a substantial amount of bile in bilirubin excretion
and can therefore be of use in diagnosing cholestasis.
DRUG METABOLISM
Cytochrome P450 is responsible for the oxidation of many xenobiotics.
It usually involves two phases:
◦ Phase I reactions - oxidations/hydroxylations
◦ Phase II reactions (conjugation) - conjugate the metabolite to polar
compounds such as glucuronic acid, glycine, taurine, and sulfate.
SYNTHETIC FUNCTIONS
PROTEIN SYNTHESIS
◦ Destruction of liver tissue will result in low serum levels of total protein and
albumin.
◦ Biuret method is used to determine serum protein levels.
◦ The reference range for total serum protein levels is generally in the 6-7.8
g/dLrange
ALBUMIN
It is the major protein synthesized in the liver

Normal albumin synthesis is about 120 mg/kg/day.

OTHER SERUM PROTEINS
Alpha-1- Antitrypsin (AAT) – most abundant and most important
protease inhibitor in plasma.

Ceruloplasmin - copper-containing protein in serum. Wilson’s disease

results in low levels of ceruloplasmin.
CLOTTING FACTORS
Coagulation proteins are made in the liver

Inhibitors of coagulation are synthesized in the liver

Fibrin degradation products are catabolized in the liver

Disseminated intravascular coagulopathy (DIC) is the most common

coagulopathy seen in liver disease.
CLOTTING FACTORS
The most frequently ordered laboratory test for detecting liver-
associated coagulation abnormalities is the prothrombin time (PT).
TESTS FOR LIVER INJURY
Cellular Locations of Enzymes
◦ Cytoplasmic enzymes -lactate dehydrogenase (LD), aspartate
aminotransferase (AST), and alanine aminotransferase (ALT).
◦ Mitochondrial enzymes - mitochondrial isoenzyme of AST
◦ Canalicular enzymes - alkaline phosphatase and gamma-glutamyl transferase
(GGT)
AMINOTRANSFERASES
Aspartate aminotransferase (AST), aka serum glutamate oxaloacetate
transaminase (SGOT) – intra- and extra-mitochondrial.

Alanine aminotransferase (ALT), aka serum glutamate pyruvate

transaminase (SGPT) – extramitochondrial. Found mainly in liver.
LACTATE
DEHYDROGENASE
LD1 and LD2 predominate in cardiac muscle, kidney, and erythrocytes.
LD4 and LD5 are the major isoenzymes in liver and skeletal muscle.
The upper reference range limit for total LD activity in serum is around
150 IU/L.
Serum LD levels become elevated in hepatitis; often, these increases are
transient and return to normal by the time of clinical presentation
 LACTATE
DEHYDROGENASE
If there is the large increase of total LD to levels of 500 IU/L or more,
combined with a significant increase in alkaline phosphatase (ALP),
space-occupying lesions of the liver
CANALICULAR INJURY
Alkaline Phosphatase (ALP)
◦ It rises in conditions like obstruction of the biliary tract from stones in the
ducts or ductules, or infectious processes resulting in ascending cholangitis,
or from space-occupying lesions
◦ The measurement of this enzyme activity was suggested as one method of
discriminating intrahepatic from extrahepatic jaundice
CANALICULAR INJURY
Gamma-Glutamyl Transferase(GGT)

◦ If ALP is elevated and GGT is also elevated, then the source of the elevated
ALP is most likely biliary tract.

◦ GGT remains normal even during cholestasis in pregnancy.

◦ GGT is often increased in alcoholics even without liver disease

ALPHA-FETOPROTEIN
AFP is an important marker for hepatocellular carcinoma (HCC).

Elevated levels can also occur after acute liver disease and also fibrosis
AUTOIMMUNE MARKERS
Antimitochondrial Antibody Is a Marker for Primary Biliary Cirrhosis
(PBC)

ANCA is a Marker for Primary Sclerosing Cholangitis

Serum Markers for Autoimmune Hepatitis

 Table 21-1 -- Interpretations of Patterns of HBV Markers

HEPATITIS B
Interpretation IgM Anti- Total
HBc Anti-HBc
HBsAg Anti-
HBs
HBeAg Anti-
HBe
Incubation period of HBV - - + - - -
infection
Acute HBV infection + + + - + -
Recent, resolving HBV + + - + - +
infection
Acute HBV infection in + + - - - -
core window
Active chronic HBV - + + - + -
infection
Chronic HBV carrier state - + + - - +

Resolved HBV infection - + - + - +

HBV immunity after - - - + -

vaccination
HEPATITIS C
HCV belongs to the flavivirus group. Most cases occur in injection drug
users and accidental needle puncture.
Chronic infection occurs in 85% of infected individuals.
Chronically infected individuals will have an elevated ALT level.
There is 1-5% risk of developing HCC after 20 years in patients with
chronic HCV.
HEPATITIS C
The major diagnostic test for HCV infection has been the second-
generation anti-HCV, which detects presence of antibody to one of four
different viral antigens an average of 10-12 weeks after infection ( Alter,
1992a ).
DIAGNOSIS OF LIVER
DISEASE
HEPATITIS
◦ A rise in the aminotransferases to values of more than 200 IU/L and often to
500 or even 1000 IU/L.
◦ LD levels are mildly elevated to values typically around 300-500 IU/L due to
hepatocyte damage.
◦ Because of inflammation and/or necrosis apoptosis of canalicular and
ductular lining cells, the alkaline phosphatase may also be elevated to values
of typically 200-350 IU/L.
DIAGNOSIS OF LIVER
DISEASE
CHRONIC PASSIVE CONGESTION

◦ a mild increase in the aminotransferases and occasionally a mild

hyperbilirubinemia.
 CIRRHOSIS
◦ Compression of the intrahepatic bile ductules
DIAGNOSIS and cholangioles results in diminished
excretion of bilirubin and bile salts, causing
OF LIVER hyperbilirubinemia and a rise in alkaline
phosphatase, GGT and 5′-nucleotidase.
DISEASE ◦ The serum concentrations of hepatocyte
enzymes, like AST, ALT and LD are either normal
or diminished.
 POSTHEPATIC AND POSTHEPATOCYTIC
BILIARY OBSTRUCTION
DIAGNOSIS
OF LIVER ◦ elevated bilirubin, most of it direct, ALP and
GGT.
DISEASE
DIAGNOSIS OF LIVER
DISEASE
SPACE OCCUPYING LESION
◦ There is increase in LD and alkaline phosphatase.
Disease Diagnostic Test

Hepatitis A Anti-HAV IgM

Hepatitis B

Acute HBsAg and anti-HBc IgM

Chronic HBsAg and HBeAg and/or HBV DNA

Hepatitis C Anti-HCV and HCV RNA

Hepatitis D (delta) HBsAg and anti-HDV

Hepatitis E Anti-HEV

Autoimmune hepatitis ANA or SMA, elevated IgG levels, and

compatible histology
Primary biliary cirrhosis Mitochondrial antibody, elevated IgM
levels, and compatible histology
Primary sclerosing cholangitis
Drug-induced liver disease
Alcoholic liver disease
Nonalcoholic steatohepatitis
α1 Antitrypsin disease
Wilson disease
Hemochromatosis
Hepatocellular cancer
P-ANCA, cholangiography
History of drug ingestion
History of excessive alcohol intake and
compatible histology
Ultrasound or CT evidence of fatty liver and
compatible histology
Reduced 1 antitrypsin levels, phenotypes PiZZ
or PiSZ
Decreased serum ceruloplasmin and increased
urinary copper; increased hepatic copper level
Elevated iron saturation and serum ferritin;
genetic testing for HFE gene mutations
Elevated -fetoprotein level >500; ultrasound
or CT image of mass
Markers of pancreatic disease
Acute Pancreatitis

Serum amylase: universal laboratory diagnostic test in the

determination of acute pancreatitis
Derived from pancreatic acinar cells
Rises over the first 2-12 hours after the onset of acute pancreatitis,
peaks at 48 hours and returns to normal within 3-5 days
Acute Pancreatitis

new-onset sharp, ‘boring’ epigastric pain radiating to back or flanks

associated with nausea and vomiting

serum amylase helps to confirm the suspected diagnosis of acute

pancreatitis
Acute Pancreatitis

The specificity is limited by elevations in amylase from inflammatory

intra-abdominal processes, parotid and submandibular salivary gland
inflammation
Acute Pancreatitis

Serum lipase rises slightly earlier than amylase, 4-8

hours after the onset of acute pancreatitis, and peaks
earlier, at 24 hours. The serum lipase also lasts longer
in the serum, 8-14 days. For these reasons, serum
lipase is more sensitive and specific than the serum
amylase.
However, the utility of serum lipase in acute
pancreatitis has been shown to vary due to
discrepancies in measurement method, patient
selection, and cutoff point
Acute Pancreatitis

The use of a serum lipase in the diagnosis of acute pancreatitis should

be reserved to patients with clinical symptoms consistent with the
disease and an amylase that is suspected to be falsely low (such as
in alcoholics, patients with hypertriglyceridemia, or
presenting late with the disease)
Acute Pancreatitis

utilizing serum lipase in conjunction with serum amylase as a routine

process in the laboratory evaluation of suspected acute pancreatitis
should be considered inappropriate.
 plasma calcitonin precursors
determination of severity of an acute episode of pancreatitis.
Abnormal levels can be detected usually within hours after the onset of
abdominal pain.
To distinguish etiology
ALT and/or AST of more than 150 IU/dL (a threefold elevation) had a
positive predictive value of 95% in predicting gallstones as the
underlying cause.
Bilirubin and alkaline phosphatase have a limited role in the
diagnosis of gallstone acute pancreatitis.
Inflamed pancreas: gallstone
pancreatitis

Damaged pancreas over the

years of alc consumption:
alcohol-induced pancreatitis
• -The ducts have been altered by the
deposition of proteinaceous plugs.
The gland has altered architecture
and becomes ‘burned out.’
• -Amylase is affected, the lipase is not
as affected.
 appears useful in the
determination of
alcoholism.
carbohydrate
deficient
transferrin
(CDT) an ideal marker in a patient
suspected of being an
alcoholic, who denies
alcohol use when the
alcohol level is normal.
Detection of trypsinogen activation peptide (TAP)
Severity Hematocrit
Detection of Severity
TRYPSINOGEN ACTIVATION PEPTIDE
(TAP)
 Inappropriate early activation of
trypsin in the acini of the pancreas
leads to the release of TAP. In
patients with acute pancreatitis,
TAP levels rise. A TAP >30
mmol/L: associated with severe
disease, with a negative predictive
value of 100%
HEMATOCRIT
Hct above 44 or rising over the first
24: associated with pancreatic
necrosis. This is likely related to
hemoconcentration from a
combination of severe third space
losses, fluid sequestration, and poor
intravenous hydration.
A serum C-reactive protein is
useful later (after 36-48 hours
after the onset of symptoms)
in determining the presence
of pancreatic necrosis
  characterized by abnormal secretion from the
various exocrine glands of the body, including

-pancreas
-salivary glands,

Cystic -peritracheal,

Fibrosis -peribronchial, and

-peribronchiolar glands,
-lacrimal glands,
- sweat glands
-mucosal glands of the small bowel, and bile
ducts.
Cystic Fibrosis
Involvement of the intestinal glands may result in the presence of
meconium ileus at birth. Chronic lung disease and malabsorption
resulting from pancreatic involvement are the major clinical problems
of those who survive beyond infancy.
Cystic Fibrosis
laboratory diagnosis still depends largely on the demonstration of
increased sodium and chloride in the sweat.

In children, [Cl] >60 mmol/L of sweat on at least two occasions are

diagnostic,; between 50-60 mmol/L are suggestive in the absence of
adrenal insufficiency.

Sweat chloride concentrations of more than 60 mmol/L:

• malnutrition, hyperhidrotic ectodermal dysplasia, nephrogenic diabetes insipidus, renal

insufficiency, G6PD, hypothyroidism, mucopolysaccharidosis, and fucosidosis.
Cystic Fibrosis
[Na] in sweat slightly lower than those of
chloride in patients with cystic fibrosis, but
the reverse is true in normal subjects.

False-negative sweat test results have been

seen in patients with cystic fibrosis in the
presence of hypoproteinemic edema.
Cystic Fibrosis
Premenopausal women: cyclic fluctuation, reaching a peak chloride
concentration most commonly 5-10 days prior to the onset of menses.
Peak values were slightly under 65 mmol/L.
Men: random fluctuations up to 70 mEq/L.
END