Liver Function Tests

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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
Notes on Liver function tests.. By Dr. Ashish Jawarkar Contact: pathologybasics@gmail.com Web: pathologybasics.wix.com/notes
* 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
(i) BILIRUBIN:
Serum Bilirubin Types: Indirect Bilirubin (unconjugated) 90% more of total 1. Tightly bound to albumin 2. water insoluble 3. not excreted in urine
#
Direct Bilirubin (conjugated) 10% or less of total 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 + accelerator
Serum + diazo reagent
Pink Azobilirubin + alkaline tartarate Blue azobilirubin
Pink azobilirubin +alkaline tartarate Blue azobilirubin
Measure absorbance at 600nm
Measure absorbance at 600 nm
Total bilirubin
Direct bilirubin
Indirect bilirubin = total bilirubin direct bilirubin
Normal Levels: Total Bilirubin Direct Bilirubin Patterns: Normal Post hepatic type Hepatic type Pre hepatic type Direct 10% of total Direct >50% of total Direct 20-50% of total Direct <15% of total 0.3-1.0 mg/dl 0 0.2 mg/dl
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. Gmelins 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
3. Lugols iodine 4 ml lugols iodine in test tube + 4 drops urine shake Green color indicates positive taste 4. Fouchets test 5 ml urine + 2.5 ml 10% BaCl2
Look for ppt formation
Obtain ppt on filter paper
Add 1 drop fouchets 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 Bilirubin Urobilinogen Prehepatic Absent Increased Hepatic Present Increased Post Hepatic Present Absent
(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. Ehrlichs aldehyde test
5 ml urine + 0.5 ml Ehrlichs 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 Ehrlichs reagent, BaCl2 is added and ppt is removed, which removes the bilirubin and test is performed on the filterate.
If Porphobilinogen is suspected Watson-Shwartz test is performed 5ml urine + 0.5 ml Ehrlichs 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 Porphobilinogen suspected
Chloroform layer Urobilinogen confirmed
Decant pink acqueous solution
Add butanol
Pink color in butanol layer Butanol layer Acqueous layer
Indicates porphobilinogen
2. Reagent strip method On reagent strips, test area is impregnated with p-dimethylaminobenzal dehyde or 4methoxy benzene tetrafluoroborate
Normal levels: Normal Increased urobilinogen in urine Decreased urobilinogen in urine 0.5-4mg in 24 hours 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
(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
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(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
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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)
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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:
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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
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.
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Normal Abnormal
Alb
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 1
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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 1
Chronic Inflammation -Immunoglobulin synthesis by antigen activated B lymphocytes transformed to plasma cells is demonstrated by the increased polyclonal gamma band.
Normal Abnormal
Alb
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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
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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.
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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.
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(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
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(vi) Serum aminotransferases (SGOT (AST) & SGPT (ALT))

ALT (cytosol)
GGT Canalicular surface and microsomes
Canalicular surface
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 ALT / SGPT AST:ALT
5-40 U/L 5-42 U/L 0.7-1.4
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Patterns: >15 times increase 1. 2. 3. 1. 2. 3. 4. Acute viral hepatitis Toxin induced hepatocellular damage (CC l4) Centrilobular necrosis due to ischemia (like CCF) Chronic hepatitis Autoimmune hepatitis Alcoholic hepatitis Drug induced hepatitis Recovery from acute hepatitis Vs Massive liver necrosis
5-15 times increase
Gradual decrease in enzymes
Hepatocellular jaundice AST and ALT increase >500 U/L
Cholestatic jaundice AST and ALT increase <200 U/L
Alcoholic hepatitis AST:ALT > 2 AST:ALT <1
Acute viral hepatitis
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(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 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) Normal Levels: ALP Males: 25-120 U/L Females: 25-90 U/L Bone diseases 1. Active bone growth in children 2. Osteomalacia 3. rickets 4. Hyperparathyroidism 5. Pagets 6. Osteosarcoma 7. Osteoblastic metastasis Pregnancy 1. Placental ALP
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(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: Females: upto 40U/L upto 25U/L
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(ix) 5 Nucleotidase
a. Is released from liver only b. Used to know whether raised ALP is from liver or other sources
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(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 Normal value (>50% excreted in bile) High (<50% excreted in bile) @2 hr in blood Higher than expected (slow excretion after 45 min) Lower than expected (Fast excretion after 45 min)
Dubin Johnson syndrome Rotor syndrome
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*INTERPRETATION OF LIVER FUNCTION TESTS Typical LFT values in: Hepatocellular diseases Hyperbilirubinemia Unconjugated>conjugated Ie indirect>direct Conjugated 20-50% of total AST and ALT - >500 IU/L ALP raised <3 times Usually no increase in GGT, 5NT Cholestatic diseases Hyperbilirubinemia Conjugated>unconjugated Ie direct>indirect Conjugated >50% of total AST, ALT- 200-500 IU/L ALP raised >3 times elevation of GGT and 5NT
1.
1.
2. 3. 4.
2. 3. 4.
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*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 AST:ALT>2
Toxic or ischemic injury AST,ALT>3000IU/L
Drug induced
Acute hepatitis A IgM Anti HAV
Acute Hepatitis B IgM anti HBcAg HBsAg
Acute hepatitis C Anti HCV HCV RNA
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*APPROACH TO SUSPECTED CHOLESTATIC DISEASE Suspected cholestatic jaundice (High direct bilirubin, clinically)
Raised ALP
Evaluate GGT and 5NT
Raised GGT, 5NT
Normal GGT, 5NT
Proves hepatic cause Of raised ALP
Non hepatic cause of Raised ALP
Abdominal USG/CT
Dilatation of bile ducts
No dilatation of bile ducts
Extrahepatic cause
Intrahepatic cause
Radiology
Radiology
a. b. c. d.
PSC Ca head of pancreas Stricture Stone in CBD
Liver mass
Diffuse Liver disease
FNAC
Biopsy
1 or 2 Ca
Infilatrative liver dis 1 biliary cirrhosis

Liver Function Tests

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Clinical pathology notes LIVER FUNCTION TESTS

Method (Di azo method):

Serum + di azo reagent + accelerator

Serum + diazo reagent

Pink Azobilirubin + alkaline tartarate Blue azobilirubin

Pink azobilirubin +alkaline tartarate Blue azobilirubin

Measure absorbance at 600nm

Measure absorbance at 600 nm

Indirect bilirubin = total bilirubin direct bilirubin

Play of colors from yellow to violet to blue to green

Look for ppt formation

Obtain ppt on filter paper

Add 1 drop fouchets reagent

Blue green color immediately

(ii) URINE UROBILINOGEN

Bile contains conjugated bilirubin

Converted by bacterial action in intestines to urobilinogen

Some urobilinogen not taken up by liver is excreted in urine

5 ml urine + 0.5 ml Ehrlichs aldehyde reagent 5 min, room temp

Dark red color

Pink / Dark red solution

Add chloroform 1-2 ml

Pink color in aqueous layer

Pink color in chloroform layer

Chloroform layer Porphobilinogen suspected

Chloroform layer Urobilinogen confirmed

Decant pink acqueous solution

Pink color in butanol layer Butanol layer Acqueous layer

(iii) Prothrombin time

(iv) Serum Proteins

Violet color compound

Color intensity read by colorimeter Normal: Total Protein 5.5 8 gm/dl

Measured colorimetrically Normal Values: Serum albumin

3.5 5 gm/dl (60% of total proteins)

4. Serum Protein electrophoresis Following pattern can be observed on electrophoresis: Normal:

Serum IFE in monoclonal gammopathy

Urine IFE in monoclonal gammopathy

(v) Blood Ammonia Level

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

GGT Canalicular surface and microsomes

AST (mito and cytosol)

Location of liver enzymes

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

Levels correlate with extent of tissue damage Normal levels:

AST / SGOT ALT / SGPT AST:ALT

5-40 U/L 5-42 U/L 0.7-1.4

5-15 times increase

Gradual decrease in enzymes

Hepatocellular jaundice AST and ALT increase >500 U/L

Cholestatic jaundice AST and ALT increase <200 U/L

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

Acute viral hepatitis

(vii) Serum Alkaline Phosphatase (ALP)

Hence diseases that affect hepatocyte secretion have elevated ALPs

It is used primarily to differentiate between hepatocellular and cholestatic jaundice

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

(viii) Serum GGT

(x) Dye Excretion test

Check for dye in blood at 45 min

If >50% retained liver function is abnormal

Dubin Johnson syndrome Rotor syndrome

AST, ALT raised - >300IU/L