Liver function tests

this is a series of notes on clinical pathology, useful for undergraduate and post graduate pathology students. Notes have been prepared from standard textbooks and are in a format easy to reproduce in exams.

1. Clinical pathology notes
LIVER FUNCTION TESTS
by Dr. Ashish Jawarkar (M.D. Pathology)
Consultant Pathologist
Parul Sevashram Hospital
Vadodara



OVERVIEW
1. Indications of LFT
2. Limitations of LFT
3. Classification of LFTs
a. tests that assess excretory function
i. bilirubin in serum and urine
ii. urobilinogen in urine and feces
b. tests that assess synthetic function
i. serum protein
ii. serum albumin
iii. serum albumin:serum globulin ratio
iv. Prothrombin time
v. Serum protein electrophoresis
c. tests that assess metabolic function
i. Blood ammonia level
d. tests that assess hepatic injury
i. ALT/SGPT
ii. AST/SGOT
iii. Alkaline phosphatase
iv. Gamma GGT
v. 5-nucleotidase
e. tests that assess clearance of exogenous substances
i. Bromsulphathelien excretion test
4. Each LFT in detail
5. Interpretation of LFT
6. Approach to a patient with suspected hepatocellular disorder, cholestatic disorder

2. * INDICATIONS OF LIVER FUNCTION TESTS

1. Screening of suspected liver disorder
2. to find out type of liver disease
a. hepatocellular
b. cholestatic
c. infiltrative
3. assess the severity and prognosis of liver disease
4. follow up the course of liver disease through the recovery phase

* LIMITATIONS OF LIVER FUNCTION TESTS

1. Lack sensitivity
Liver has large anatomic and functional reserve; there has to be extensive liver damage
for LFTs to derange

2. Lack specificity
LFTs are abnormal in various non hepatic conditions:
a. raised bilirubin
i. hemolysis
ii. ineffective erythropoeisis
iii. large hematoma
b. raised aminotransferases
i. muscle injury
ii. alcohol abuse
iii. MI
c. raised alkaline phosphatase
i. pregnancy
ii. bone disorders
d. low serum albumin
i. poor nutrition
ii. proteinuria
iii. malabsorption
iv. severe illness causing catabolism

3. (i) BILIRUBIN:

Serum Bilirubin

Types:

Indirect Bilirubin (unconjugated)
90% more of total
Direct Bilirubin (conjugated)
10% or less of total
1. Tightly bound to albumin
2. water insoluble
3. not excreted in urine
1. includes bilirubin glucoronide, bilirubin
diglucoronide and delta bilirubin
#

2. water soluble
3. can be excreted in urine

#
consists of conjugated bilirubin bound to
albumin, level is increased in cholestasis,
excreted slowly in urine

Method (Di azo method):


Serum + di azo reagent Serum + diazo reagent

+ accelerator


Pink Azobilirubin Pink azobilirubin

+ alkaline tartarate +alkaline tartarate

Blue azobilirubin Blue azobilirubin




Measure absorbance at 600nm Measure absorbance at 600 nm




Total bilirubin Direct bilirubin


Indirect bilirubin = total bilirubin – direct bilirubin

4. Normal Levels:

Total Bilirubin 0.3-1.0 mg/dl
Direct Bilirubin 0 – 0.2 mg/dl

Patterns:

Normal Direct 10% of total
Post hepatic type Direct >50% of total
Hepatic type Direct 20-50% of total
Pre hepatic type Direct <15% of total


Urine bilirubin

Rationale:

1. Presence of bilirubin in urine indicates conjugated hyperbilirubinemia due to obstructive
or hepatocellular causes.
2. Bilirubin is absent in urine in hemolytic jaundice because unconjugated bilirubin is not
soluble in water.

Methods:

1. Foam test

5 ml urine in test tube

shake
Yellow foam


Bilirubin present

2. Gmelin’s test

3 ml conc nitric acid in test tube + pour 3 ml urine slowly over it


Play of colors from yellow to violet to blue to green


Positive test

5. 3. Lugol’s iodine

4 ml lugol’s iodine in test tube + 4 drops urine

shake

Green color indicates positive taste

4. Fouchet’s test

5 ml urine + 2.5 ml 10% BaCl2


Look for ppt formation


Obtain ppt on filter paper


Add 1 drop fouchet’s reagent


Blue green color immediately


Bilirubin is present


5. Reagent strips impregnated with diazo reagent

Can detect minimum 0.5 mg/dl of bilirubin

Patterns:

Urine Prehepatic Hepatic Post Hepatic
Bilirubin Absent Present Present
Urobilinogen Increased Increased Absent

6. (ii) URINE UROBILINOGEN

Rationale:


Bile contains conjugated bilirubin



Converted by bacterial action in intestines to urobilinogen



Enterohepatic circulation



Some urobilinogen not taken up by liver is excreted in urine


On exposure to air (urine), urobilinogen is converted to urobilin which gives urine its pale yellow
color

Method:

1. Ehrlich’s aldehyde test


5 ml urine + 0.5 ml Ehrlich’s aldehyde reagent
5 min, room temp



Pink color Dark red color


Normal urobilinogen Increased urobilinogen

Fallacy:
This test is positive with urobilinogen, bilirubin and porphobilinogen.

If bilirubin is suspected; before adding Ehrlich’s reagent, BaCl2 is added and ppt is
removed, which removes the bilirubin and test is performed on the filterate.

7. If Porphobilinogen is suspected – Watson-Shwartz test is performed

5ml urine + 0.5 ml Ehrlich’s aldehde reagent



Pink / Dark red solution



Add chloroform 1-2 ml






Pink color in aqueous layer Pink color in chloroform layer



Acqueous layer acqueous layer


Chloroform layer Chloroform layer

Porphobilinogen suspected Urobilinogen confirmed



Decant pink acqueous solution


Add butanol


Pink color in butanol layer

Butanol layer

Acqueous layer


Indicates porphobilinogen

8. 2. Reagent strip method

On reagent strips, test area is impregnated with p-dimethylaminobenzal dehyde or 4-
methoxy benzene tetrafluoroborate


Normal levels:

Normal 0.5-4mg in 24 hours
Increased urobilinogen in urine

Decreased urobilinogen in urine
Hemolytic jaundice, hemorrhage in tissues

Obstructive jaundice, reduction of intestinal
flora

Fallacy:
False negative results may be obtained if -
1. UTI – oxidizes urobilinogen to urobilin
2. antibiotic therapy – eliminates gut bacteria, no urobilinogen produced

9. (iii) Prothrombin time

Rationale:
1. Most of the clotting factors (except vWF) are synthesized in the liver, hence clotting
function would be deranged in liver disorders
2. Vitamin K dependent clotting factors include factor II, VII and IX, and X. PT measures the
activity of II, VII and X.

Method:

Platelet poor plasma

+

Tissue thromboplastin (activates extrinsic pathway)

+

Calcium chloride

Allowed to clot


Note time

Causes of raised PT:
1. Hepatocellular disease
#

2. Obstructive jaundice
#
– malabsorption of vitamin K – lack of synthesis of vitamin K
dependent clotting factors
3. DIC – exhaustion of coagulation factors
4. Inherited deficiency of coagulation factors

#
To differentiate raised PT due to hepatocellular disease or obstructive causes, repeat after
administration of Vit K

Normal values:

PT 11 To 16 seconds

10. (iv) Serum Proteins

1. Total Proteins

Rationale:
1. Liver is the sole site for synthesis of all proteins except gammaglobulins (which is
synthesized by plasma cells).
2. Hence measurement of total proteins is a fair indicator of liver function
3. However, the decrease in proteins synthesized by liver is compensated by increased
synthesis of gamma globulins by plasma cells.
4. Hence overall value of total serum protein measurement is limited.

Methods:

1. Refractometer method:

Refractive index of serum is measured and read directly from the refractometer

2. Biuret method:

Copper ions in the reagent react with peptide bonds of the protein


Violet color compound


Color intensity read by colorimeter

Normal:

Total Protein 5.5 – 8 gm/dl

11. 2. Serum Albumin

Rationale:

1. Albumin consists of 60% of total proteins and is exclusively synthesized in liver. Hence
its estimation can help in liver diseases (especially chronic). Serum albumin level is low
in chronic liver diseases like cirrhosis and also correlates with severity like progression of
ascites.
2. The half life of albumin is 20 days, hence its level does not fall with acute diseases such
as acute hepatitis.
3. Serum albumin measurement is not specific because albumin also falls in
a. Malnutrition
b. Malabsorption
c. Decreased sythesis – liver disease, chronic infection
d. Increased catabolism – thyrotoxicosis, malignancies, infection
e. Increased loss
i. Nephrotic syndrome
ii. Burns
iii. Protein loosing enteropathy
f. Increased blood volume (dilution – false low)
i. Pregnancy
ii. CCF

Method:

BROMOCRESOL GREEN METHOD;

Serum + Bromo cresol green


Binds to albumin



Blue color


Measured colorimetrically

Normal Values:
Serum albumin 3.5 – 5 gm/dl (60% of total proteins)

12. 3. Serum albumin : Serum globulin ratio

Rationale:

1. The total serum plasma protein level is affected by compensatory increase of gamma
globulins as albumin falls
2. The ratio of serum albumin to globulin gives a better idea of the liver function

Normal:

Normal albumin:globulin ratio >1:5



4. Serum Protein electrophoresis

Following pattern can be observed on electrophoresis:

Normal:

13. Cirrhosis of liver:

When liver function is sufficiently diminished, protein synthesizing capacity is
compromised and concentrations of albumin and proteins in the alpha and beta bands are
decreased. An additional common finding is beta-gamma bridging due to increased IgA.








Beta-gamma bridging



Alb α1 α2 β γ


The Nephrotic Syndrome --

1. Renal disease involving the glomeruli is always associated with increased urinary protein
loss. When protein loss is greater than 3-4 g/day, the protein synthesizing capacity of
the liver is exceeded and hypoproteinemia, accompanied by anasarca, develops to
cause the nephrotic syndrome.
2. The massive urine protein loss is due to increased permeability of glomeruli to protein.
The permeability increase may be minimal so that only albumin and other smaller
molecular weight proteins are selectively filtered (selective nephrosis, as in Minimal
Change Disease) or may be greater so that larger proteins are also filtered (nonselective
nephrosis, as in membranous golmerulonephritis) as is the case in the example shown.
3. Alpha-2-macroglobulin is sufficiently large so that it is not filtered and increased
synthesis (from the general hepatic protein synthesis) causes its accumulation.
4. Lipoproteins are also sufficiently large to accumulate and hyperlipidemia is a
characteristic of the nephrotic syndrome, although lipoproteins are not stained with the
protein stain used in visualizing proteins.

14. Normal
Abnormal







Alb α1 α2 β γ



Alpha-1-Antitrypsin Deficiency:

A genetic defect causes a deficiency of alpha-1-antitrypsin. The antiprotease deficiency
results in a propensity to develop emphysema. Since alpha-1-antitrypsin is the major
component of the alpha-1 band, deficiency is suggested by a reduced alpha-1 band. Deficiency is
confirmed by specific immunochemical quantification.



Normal
Abnormal








Alb α1 α2 β γ

15. Acute Inflammation
The alpha-1 and alpha-2 bands are increased during the inflammatory response from
increased hepatic synthesis of acute phase reactant proteins.

Normal

Abnormal






Alb α1 α2 β γ



Chronic Inflammation --

Immunoglobulin synthesis by antigen activated B lymphocytes transformed to plasma
cells is demonstrated by the increased polyclonal gamma band.


Normal
Abnormal








Alb α1 α2 β γ

16. Immunoglobulin Deficiency --

Deficient immunoglobulin synthesis is revealed by a markedly diminished gamma band.
Effected individuals are prone to recurrent infection














Monoclonal Gammopathy --

1. An unusually sharp band in the gamma region strongly suggests the presence of a
homogeneous immunoglobulin and, thus, the malignant proliferation of plasma cells
from a single cell (multiple myeloma) in contrast to the broad, heterogeneous, or
polyclonal, gamma band as exhibited above in chronic inflammation from
immunoglobulin synthesis by many different clones of plasma cells.
2. Homogeneous immunoglobulins are also found in Waldenstrom's macroglobulinemia
(where the sharp gamma band is always IgM). Specimens which exhibit a narrow
gamma band are further examined by immunofixation electrophoresis as described
below











Alb α1 α2 β γ

17. ImmunoFixation Electrophoresis

Immunofixation electrophoresis (IFE) is used to demonstrate that a narrow gamma band
is due to a homogeneous immunoglobulin. IFE has superceded immunoelectrophoresis, results
from which are considerably more difficult to interpret, for the purpose of evaluating
monoclonal gammopathy.

In the illustration below, 6 replicates of the specimen are loaded on to separate lanes of
an IFE gel. Following electrophoresis, the protein in each lane is stained differently. The first lane
(SP) is stained for total protein. The protein in each of the other lanes is "stained" with specific
antisera for immunoglobulin heavy and light chains, respectively, as illustrated in the figure. The
finding of the preponderance of only one light chain associated with a predominantly staining
heavy chain confirms the molecular homogeneity of the immunoglobulin and also provides
identification. IFE identifies the narrow band as monoclonal IgG, lambda.

Sometimes malignant plasma cells synthesize excess light chains and less frequently
only light chains are synthesized. Almost never is excess heavy chain or only heavy chain
synthesized. Excess free lambda light chain is exhibited in the illustration.













Serum IFE in monoclonal gammopathy



Free light chains are readily filtered by the glomeruli and often are not detectable
in serum specimens. Excess light chain synthesis results in proteinuria, which exhibits a
narrow band upon electrophoresis. The identity of the narrow band is determined by IFE
and is here seen to be free lambda light chain. (A trace of monoclonal IgG,lambda is also
present in the urine specimen). The bottom-most band is albumin.

18. Urine IFE in monoclonal gammopathy


Polyclonal Gammopathy.

An IFE of a serum specimen with a polyclonal gamma increase is shown for the sake of
comparison.

Serum IFE

Broad bands are present for both IgG and IgA and corresponding kappa and lambda light chain
bands. The light chains appear to be present at the normal kappa/lambda ratio of about 2.

19. (v) Blood Ammonia Level

Rationale:
1. Ammonia is detoxified and converted to ammonia in liver, estimation of blood ammonia
is an indicator of metabolic function of the liver.
2. Estimation of blood ammonia helps in knowing the cause in patients of coma of
unknown origin
3. Very nonspecific because it is also raised in
a. Reyes syndrome
b. Shunting of portal to systemic blood
c. GI hemorrhage – increased production of ammonia
d. Inherited deficiency of urea cycle enzymes

Normal:

Ammonia 9-33 micromol/l

20. (vi) Serum aminotransferases (SGOT (AST) & SGPT (ALT))

ALT (cytosol)





GGT

Canalicular surface
Canalicular surface and microsomes


5’ nucleotidase
ALP




LDH (cytosol) AST (mito and cytosol)

Location of liver enzymes


Rationale:
Normally these enzymes are present in serum at low levels




When necrosis or cell death occurs, these are released into the blood



Levels correlate with extent of tissue damage

Normal levels:


AST / SGOT 5-40 U/L
ALT / SGPT 5-42 U/L
AST:ALT 0.7-1.4

21. Patterns:

>15 times increase 1. Acute viral hepatitis
2. Toxin induced hepatocellular damage (CC l4)
3. Centrilobular necrosis due to ischemia (like CCF)
5-15 times increase 1. Chronic hepatitis
2. Autoimmune hepatitis
3. Alcoholic hepatitis
4. Drug induced hepatitis
Gradual decrease in enzymes Recovery from acute hepatitis
Vs
Massive liver necrosis


Hepatocellular jaundice Cholestatic jaundice
AST and ALT increase >500 U/L AST and ALT increase <200 U/L


Alcoholic hepatitis Acute viral hepatitis
AST:ALT > 2 AST:ALT <1

22. (vii) Serum Alkaline Phosphatase (ALP)

Rationale:

1. Alkaline phosphatase is present in canalicular surface of hepatocytes


Hence diseases that affect hepatocyte secretion have elevated ALPs


It is used primarily to differentiate between hepatocellular and cholestatic jaundice



No increased ALP Increased ALP


2. It is non specific – also secreted from
a. bones
b. intestine
c. kidneys
d. placenta


Causes of raised ALP:

Cholestasis
Increased >3 times
Bone diseases Pregnancy
1. Bile duct obstruction
a. Ca head pancreas
b. Bile duct strictures
c. Biliary atresia
2. Biliary cirrhosis
3. PSC
4. infiltrative diseases of liver
(granulomas, amyloid cysts)
1. Active bone growth in
children
2. Osteomalacia
3. rickets
4. Hyperparathyroidism
5. Paget’s
6. Osteosarcoma
7. Osteoblastic metastasis
1. Placental ALP

Normal Levels:

ALP Males: 25-120 U/L
Females: 25-90 U/L

23. (viii) Serum GGT

High levels also present in diseases of (organs with ducts)
- Pancreas
- Kidney
- Prostate

Causes of raised GGT:

1. Alcoholism –
a. Marked elevation occurs in acute alcoholic hepatitis
b. Also helpful in diagnosing occult alcoholism (with no notable liver damage)

2. Cholestasis –
a. Level parallels ALP and 5’ Nucleotidase
b. This enzyme is very helpful in combination with above two

3. Recovery from acute hepatitis –
a. This is the last enzyme to return to normal following acute hepatitis –
decrease in level indicates favourable outcome

Normal levels:

GGT Males: upto 40U/L
Females: upto 25U/L

24. (ix) 5’ Nucleotidase

a. Is released from liver only
b. Used to know whether raised ALP is from liver or other sources

25. (x) Dye Excretion test

Rationale:
a. Some synthetic dyes when introduced in the body are excreted in bile
b. Their clearance depends on – hepatic circulation, hepatocyte function, uninterrupted bile
flow
c. These are sensitive tests for liver function but not used nowadays because of risk of adverse
drug reactions
d. Most common dyes used include
1. Bromsulphathlein (BSP)
2. Indocyanine green
3. Rose bengal

BSP Excretion test:
BSP injected IV


Taken up by hepatocytes


Conjugated to glutathione


Excreted in bile


Check for dye in blood at 45 min


If >50% retained – liver function is abnormal


Application:

@45 min in blood @2 hr in blood
Dubin Johnson syndrome Normal value
(>50% excreted in bile)
Higher than expected
(slow excretion after 45 min)
Rotor syndrome High
(<50% excreted in bile)
Lower than expected
(Fast excretion after 45 min)

26. *INTERPRETATION OF LIVER FUNCTION TESTS

Typical LFT values in:

Hepatocellular diseases Cholestatic diseases
1. Hyperbilirubinemia
Unconjugated>conjugated
Ie indirect>direct
Conjugated 20-50% of total
2. AST and ALT - >500 IU/L
3. ALP – raised <3 times
4. Usually no increase in GGT, 5’NT
1. Hyperbilirubinemia
Conjugated>unconjugated
Ie direct>indirect
Conjugated >50% of total
2. AST, ALT- 200-500 IU/L
3. ALP – raised >3 times
4. elevation of GGT and 5’NT

27. *APPROACH TO SUSPECTED HEPATOCELLULAR DISEASE:

Suspected hepatocellular disease (Higher indirect Bilirubin, clinically)


AST, ALT raised - >300IU/L


Acute hepatic injury



Acute Viral hepatitis Alcoholic hepatitis Toxic or ischemic injury Drug induced
AST:ALT>2 AST,ALT>3000IU/L




Acute hepatitis A Acute Hepatitis B Acute hepatitis C

IgM Anti HAV IgM anti HBcAg Anti HCV
HBsAg HCV RNA

28. *APPROACH TO SUSPECTED CHOLESTATIC DISEASE
Suspected cholestatic jaundice (High direct bilirubin, clinically)



Raised ALP


Evaluate GGT and 5’NT



Raised GGT, 5’NT Normal GGT, 5’NT




Proves hepatic cause Non hepatic cause of
Of raised ALP Raised ALP



Abdominal USG/CT



Dilatation of bile ducts No dilatation of bile ducts


Extrahepatic cause Intrahepatic cause



Radiology Radiology


a. PSC
b. Ca head of pancreas
c. Stricture Liver mass Diffuse Liver disease
d. Stone in CBD

FNAC Biopsy


1’ or 2’ Ca Infilatrative liver dis
1’ biliary cirrhosis