Leucine aminopeptidase (LAP) Test – Principle, Procedure, Results

Leucine aminopeptidase (LAP) test is a biochemical diagnostic test used to detect the activity of leucine aminopeptidase enzyme. It is an enzyme that is present in various human tissues especially liver and also in many bacteria. In clinical practice this test is performed to measure LAP levels in serum or urine in order to assess liver function. It is mainly used in the diagnosis of conditions like cholestasis obstruction of bile flow hepatitis liver cirrhosis and liver tumors. It is important because raised LAP level helps in confirming that increased alkaline phosphatase is of hepatic origin and not related to bone disorders as LAP level usually remains normal in bone diseases.

In microbiology the LAP test is used as an identification test for certain gram positive cocci. It is the process in which bacterial colonies are tested for the presence of LAP enzyme. In this test leucine-β-naphthylamide is used as a substrate which is hydrolysed by LAP producing β-naphthylamine. When reagent containing p-dimethylaminocinnamaldehyde is added a red or pink colour is produced indicating a positive test. Absence of colour change or slight yellow colour indicates negative result. This test is helpful in differentiating LAP positive organisms like Streptococcus Enterococcus and Pediococcus from LAP negative organisms such as Leuconostoc and Aerococcus.

Principle of Leucine aminopeptidase (LAP) Test

The principle of Leucine aminopeptidase (LAP) test is based on the detection of leucine aminopeptidase enzyme produced by certain microorganisms. It is the process in which the enzyme hydrolyses a specific synthetic substrate. Leucine aminopeptidase is a metallopeptidase enzyme which catalyzes the removal of leucine residue from the N-terminal end of polypeptides.

In this test a filter paper disc is impregnated with L-leucine-β-naphthylamide which acts as the substrate. When bacterial cells producing LAP enzyme are applied on the disc the substrate is hydrolysed into free leucine and β-naphthylamine. This reaction is colourless and cannot be observed directly.

In the next step a chromogenic reagent containing p-dimethylaminocinnamaldehyde is added. The liberated β-naphthylamine reacts with this reagent to form a coloured complex. The development of red bright pink or reddish-purple colour indicates a positive LAP test while absence of colour change indicates a negative reaction.

Objectives of Leucine aminopeptidase (LAP) Test

• To detect the production of leucine aminopeptidase enzyme by bacterial isolates.
• To differentiate catalase-negative gram-positive cocci on the basis of LAP enzyme activity.
• To distinguish Streptococcus Enterococcus Pediococcus and Lactococcus (LAP positive) from Leuconostoc and Aerococcus (LAP negative).
• To use as a preliminary screening test for Streptococcus-like organisms along with other biochemical tests such as PYR test.
• To evaluate hepatobiliary function by estimating LAP activity in serum or urine samples.
• To help in diagnosis of cholestasis and obstruction of bile flow.
• To differentiate the source of elevated alkaline phosphatase level whether hepatic or bone related.
• To aid in diagnosis of liver disorders such as hepatitis cirrhosis liver necrosis ischemia and hepatic malignancies.

Requirements for Leucine aminopeptidase (LAP) Test

For microbiological identification

• LAP disk containing leucine-β-naphthylamide substrate.
• Developing reagent (cinnamaldehyde reagent or DMACA).
• Pure culture of test organism (18–24 hours old).
• Sterile distilled or deionized water.
• Sterile wooden applicator stick or plastic inoculating loop.
• Clean microscope slide or sterile petri dish.
• The organism should be gram positive coccus and catalase negative.
• Quality control organisms –
  • Enterococcus faecalis (positive control).
  • Aerococcus viridans (negative control).

For clinical pathology

• Blood serum or 24-hour urine sample.
• Patient should be fasting for about 8 hours before sample collection.
• Buffer solution (phosphate buffer).
• Substrate solution (L-leucyl-β-naphthylamide).
• Colour developing reagent.
• ELISA kit components if automated method is used.

Procedure of Leucine Aminopeptidase (LAP) Test

1. A LAP disk is aseptically removed and placed on a clean microscope slide or sterile petri dish and allowed to attain room temperature.
2. The disk is moistened with small amount of sterile distilled water or reagent grade water. The disk should not be over moistened as excess water may dilute the enzyme.
3. A heavy inoculum is taken from a pure 18–24 hours culture using sterile wooden stick or plastic loop.
4. The bacterial growth is rubbed on a small area of the disk until a visible paste is formed.
5. The inoculated disk is kept at room temperature for 5–10 minutes.
6. After incubation one to two drops of developing reagent (cinnamaldehyde reagent or DMACA) is added on the disk.
7. The disk is observed within 1–3 minutes for colour development.

Result of Leucine Aminopeptidase (LAP) Test

Positive Result

• Appearance – A distinct deep red bright pink or reddish-purple colour is developed on the disk within minutes after addition of reagent.
• Interpretation – The organism produces leucine aminopeptidase enzyme.
• Common organisms are –
  • Streptococcus species (including S. pneumoniae and S. pyogenes).
  • Enterococcus species (e.g. E. faecalis).
  • Pediococcus species.
  • Lactococcus species.

Negative Result

• Appearance – No colour change is observed or slight yellow orange or salmon colour is developed on the disk.
• Interpretation – The organism does not produce leucine aminopeptidase enzyme.
• Common organisms are –
  • Leuconostoc species.
  • Aerococcus viridans.
  • Globicatella species.
  • Helcococcus species.

List of organisms in Leucine Aminopeptidase (LAP) Test

LAP Positive organisms

• Streptococcus species (S. pneumoniae S. pyogenes S. agalactiae and viridans group).
• Enterococcus species (E. faecalis and E. faecium).
• Pediococcus species.
• Lactococcus species.
• Aerococcus urinae.
• Gemella species (G. morbillorum usually positive G. haemolysans may be variable).
• Abiotrophia species.
• Granulicatella species.
• Alloiococcus otitidis.
• Dolosigranulum pigrum.
• Facklamia species.
• Ignavigranum ruoffiae.
• Vagococcus species.

LAP Negative organisms

• Leuconostoc species.
• Aerococcus viridans.
• Globicatella sanguinis.
• Helcococcus species.
• Dolosicoccus species.

Precautions of Leucine Aminopeptidase (LAP) Test

• A heavy inoculum should be used for the test as light inoculum may give false negative result.
• The LAP disk should be moistened slightly and should not be over moistened.
• Excess water on the disk may dilute the enzyme and affect colour development.
• The test should be performed using young pure culture of 18–24 hours only.
• Old cultures may lose enzyme activity and give incorrect result.
• The organism should be confirmed as gram positive coccus and catalase negative before performing the test.
• LAP disks and reagents should be stored properly as recommended.
• The developing reagent should be protected from light and air.
• Disks and reagents should be brought to room temperature before use.
• Proper aseptic technique should be followed throughout the procedure.
• In clinical samples hemolysed specimens should be avoided.
• Certain drugs may influence the test result and should be considered during interpretation.

Quality control organisms of Leucine Aminopeptidase (LAP) Test

Positive control– Enterococcus faecalis (ATCC 29212) – Positive reaction with development of deep red or bright pink colour on the disk.

Negative control– Aerococcus viridans (ATCC 11563) – Negative reaction with no colour change or slight yellow colour on the disk.

Uses of Leucine Aminopeptidase (LAP) Test

• It is used for differentiation of catalase-negative gram positive cocci.
• It helps in identification of Streptococcus Enterococcus and Pediococcus species from Leuconostoc and Aerococcus viridans.
• It is used in routine microbiological laboratories for preliminary grouping of streptococcus-like organisms.
• It helps in identification of some rare and unusual gram positive cocci such as Globicatella and Facklamia species.
• It is useful in detection of vancomycin resistant organisms like Leuconostoc and Pediococcus.
• It is used in clinical diagnosis to assess liver function and hepatobiliary disorders.
• It helps in differentiation of liver origin alkaline phosphatase from bone origin.
• It is used in diagnosis of cholestasis and biliary obstruction.
• It is useful in diagnosis of liver diseases such as hepatitis cirrhosis and liver necrosis.
• It is also used in detection of hepatic and pancreatic tumors.
• In research and industry it is used for protein and peptide analysis.
• It is used in food industry for removal of bitterness from protein hydrolysates.

Advantages of Leucine Aminopeptidase (LAP) Test

• It gives rapid results and the reaction is observed within few minutes.
• The test is simple to perform and does not require complex equipment.
• It helps in clear differentiation of catalase-negative gram positive cocci.
• It is useful in separating Streptococcus Enterococcus and Pediococcus from Leuconostoc and Aerococcus.
• It helps in early identification of vancomycin resistant organisms.
• It supports appropriate selection of antibiotics and avoids misuse of vancomycin.
• It is a cost effective test for routine laboratory use.
• It is useful in confirmation of doubtful or automated identification results.
• It helps in determining liver origin of increased alkaline phosphatase levels.
• The test is reliable when performed with fresh culture.

Limitations of Leucine Aminopeptidase (LAP) Test

• The test is applicable only for catalase-negative gram positive cocci.
• It cannot be used for gram negative organisms and catalase positive bacteria like Staphylococcus.
• A light or insufficient inoculum may give false negative result.
• The disk should not be over moistened as excess water may dilute enzyme reaction.
• Old cultures may give false negative result due to loss of enzyme activity.
• The test should be performed with fresh 18–24 hours culture only.
• Deteriorated or improperly stored reagent may fail to produce colour reaction.
• The test alone is not sufficient for final identification of organism.
• It should be used along with other biochemical tests for confirmation.
• In clinical use serum hemolysis and certain drugs may interfere with results.
• The test is less commonly used for liver diagnosis due to availability of better markers.