Principle of ESR:

When an anticoagulant is added to the blood and this well mixed venous blood is placed in a vertical tube, erythrocytes tend to settle towards bottom leaving clear plasma on top. This rate of sedimentation of red blood cells in a given interval of time is called erythrocyte sedimentation rate (ESR).

As the erythrocytes sediments, in a period of one hours, 3 stages can be observed.

• Stage I: first 10 minutes
  • It is initial period of aggregation during which rouleaux are formed and the sediment rate is low
• Stage II: next 4o minutes
  • It is a period of fast setting. Sedimentation occurs at a constant rate during this period

• Stage III: next 10 minute or more
  • The sedimentation again slows as it is the final period of packing of cells at the bottom of the tube

Factors affecting ESR:

• There are several factors that affects sedimentation of erythrocytes.

• Factors that increases ESR:

  • Anemia:
    • anemia increase ESR because the change in erythrocyte-plasma ratio favors rouleaux formation.
    • Rouleaux is aggregation of RBCs together due to their discoid shape.
    • Rouleaux have a decrease surface area and accelerate ESR
  • Increase level of fibrinogen:
    • it decreases the negative charge of erythrocyte, so RBC tend to remain apart and this promotes formation of rouleaux and increase ESR
  • Immunoglobulin:
    • increase antibody level in blood increase ESR
  • Increase cholesterol level
  • Rheumatoid arthritis
  • Chronic infections
  • Carcinoma
  • Tissue destruction and other disease

• Factors that decrease ESR:

  • Defibrinigenation:
    • removal of fibrinogen decreases ESR
  • Increase albumin and lecithin in blood
  • Abnormal or sickle shape RBCs:
  • abnormal or irregular shape of RBC lower ESR
  • Congestive heart failure

Method for ESR estimation:

Westergren method for ESR estimation is widely used method. Wintrobe method is also used for ESR determination. Wintrobe tube is smaller than westergren tube

Materials required:

1. Westergren tube or wintrobe tube
2. Anticoagulant: 0.1 M sodium citrate
3. ** in modified westergren method EDTA is used as anticoagulant

Procedure for ESR estimation:

• Withdraw 4 ml of venous blood
• Mix exact 10ml of sodium citrate with 4ml of venous blood in a tube
• Invert the tube 2-3 times to mix the blood thoroughly with anticoagulant
• Fill the westergren tube up to mark 0 and place in the rack at room temperature undisturbed and away from sunlight.
• Take the reading exactly after 1 hour. Record in millimeters from top surface of column to top of RBC sediments.

Result:

• Normal value of ESR
  • Female:
    • under 50 years- 20 mm/hr
    • above 50 years- 30mm/hr
  • Male:
    • Under 50 years- 15mm/hr
    • Above 50 years- 20 mm/hr

Clinical application of ESR estimation:

• ESR test is non-specific test although it is used as indication of presence of disease
• ESR value increase during rheumatoid arthritis, chronic infection, carcinoma, tissue destruction and nephritis
• During pregnancy, ESR increase moderately from 10^th or 12^th weeks onwards and return to normal after delivery.
• ESR value decreases in sickle cell anemia and congestive heart failure (CHF).

Erythrocyte sedimentation rate (ESR): principle, method, procedure and clinical application

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Objective:

• to detect aromatic amino acid  tyrosine or histidine

Principle:

Diazotized sulphanilic acid couples with amines, phenols and imidazole to form highly colored azo compounds. This coupling reaction must be done in cold condition since diazonium compound is formed in cold. Amino acids tyrosine or histidine coupled with diazonium salt in alkaline condition to form red coloured azo dye.

Reagents:

• 1 % tyrosine, 1 % histidine, 1%glycine
• 1 % sulphanilic acid in 10 % HCI
• 5 % sodium nitrite
• 10 % sodium carbonate
• Ice bath

** Diazonium compound=Sulfanilic acid + sodium nitrite + sodium carbonate

Procedures

1. Take 2ml test solution in dry test tube.
2. Similarly, take 2ml distilled water in another test tube as control.
3. Add 1ml of sulphanilic acid, mix well and keep in ice bath.
4. Now add 1ml sodium nitrite solution to all test tubes.
5. Leave in ice bath for 3 minutes.
6. Make the solution alkaline by adding 5ml of sodium carbonate.
7. Look for the development of red colored complex.

Result interpretation:

• Positive Pauly test: red colored azo dye ( tyrosine and histidine)
• Negative Pauly test: no red color ( glycine)

Pauly’s test: Objective, Principle, Reagents, Procedure and Result

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Objective:

• to detect amino acid tryptophan present in protein

Principle:

The indole group of tryptophan reacts with glyoxylic acid in the presence of conc.  H2SO4 to give a purple colored complex.  Glyoxylic acid is prepared by reducing Oxalic acid with magnesium powder or sodium amalgam. Glacial acetic acid which has been exposed to the sunlight also contains glyoxylic acid and can thus be used for this test.

Reagents:

• 1 % tryptophan, 1 % glycine, 5 % egg white (albumin)
• Adamkevich’s reagent: glyoxylic acid ( prepared by reducing oxalic acid with magnesium powder or sodium amalgam)
• H2SO4
• Dry test tubes
• Pipettes

Procedure

1. Take 2 ml test solution in dry test tube.
2. Similarly, take 1ml distilled water in another test tube as control.
3. Add 1 ml of Adamkiewicz’s reagent, mix well.
4. Now add 1ml conc. H2SO4 along the wall of the test tube.
5. A purple colored ring develops at the interface of two solutions.

Result:

Positive Hopkin’s cole test: purple color at the interface. ( tryptophan and egg albumin)

Negative Hopkin’s cole test: glycine

Adamkiewicz reaction (Hopkin’s-cole test): Objective, Principle, Reagents, Procedure and Result

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Objective:

• to detect carbohydrate in the given solution

Principle:

Anthrone test is also another general test for all carbohydrates. In this test also, carbohydrate gets dehydrated when react with conc. H2SO4 to form furfural. This furfural reacts with anthrone to give bluish green colored complex.

Reagents:

• test solution: 5 % Glucose, 5 % Sucrose, 5 % Starch
• Anthrone reagent: 0.2 % anthrone in conc. H2SO4

Procedures

1. Take 1ml of sample in test tube.
2. Take 1ml of distilled water in another tube as control.
3. Add 2ml of anthrone reagent to all the tubes.
4. Mix thoroughly all the content of the tube.
5. Observe for color change in bluish green.

Result interpretation:

• positive test: all carbohydrate give test positive

Anthrone Test: Objective, Principle, Reagents, Procedure and Result

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Objective:

• to detect reducing sugar ( carbohydrate having free aldehyde or ketone functional group)

Principle:

The Reducing sugar under alkaline condition form enediols. Benedict’s solution contains milder alkali Na2CO3. Enediols are powerful reducing agents. They can reduce cupric ions to cuprous ions which is the basis for Benedict’s reaction. The cuprous hydroxide during the process of heating is converted to red cuprous oxide.

Reagents:

• test solutions: 5 % Glucose, 5 % Sucrose
• Benedict’s reagent: CuSO4.5H2O solution with Sodium carbonate and sodium citrate
• Water bath
• Dry test tubes
• Pipettes

Procedures

1. Take 1ml of test sample in dry test tube.
2. Take 1ml of distilled water in another tube as control.
3. Add 2ml of Benedict’s reagent to all the tubes.
4. Keep in water bath for 5 minutes.
5. Look for the development of brick red precipitate.

Result interpretation:

• positive benedict’s test: color change from blue to brick red ppt ( glucose)
• Negative Benedict’s test: no change in color( sucrose)

Benedict’s Test: Objective, Principle, Reagents, Procedure and Result

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Objective:

• to detect reducing sugar
• to distinguish monosaccharides from reducing disaccharides

Principle of Barfoed’s test:

Barfoed’s test is used for distinguishing monosaccharides from reducing disaccharides. Monosaccharides usually react in about 1-2 minute while the reducing disaccharides take much longer time between 7-12 minutes to react with the reagent. Brick red color is obtained in this test which is due to formation of cuprous oxide.

Reagents for Barfoed’s test:

• test solution: 5 % Glucose, 5 % Sucrose, 5 % Maltose, 5 % Lactose, 5 % Starch
• Barfoed’s reagent: cupric acetate in 1% acetic acid
• Water bath
• Dry test tubes
• Pipettes

Procedure of Barfoed’s test:

1. Take 1ml of test sample in dry test tube.
2. Take 1ml of distilled water in another tube as control.
3. Add 2ml of Barfoed’s reagent to all the tubes.
4. Keep in boiling water bath.
5. Look for the development of brick red precipitate.
6. Note the time taken to develop the color.

Result interpretation of Barfoed’s test:

• Positive Barfoed’s test: development of brick red color ppt within 3-5 minutes
• Negative Barfoed’s test: absence of red color

** reducing disaccharides also give positive barfoed test on prolong heating

Barfoed’s Test: Objective, Principle, Reagents, Procedure and Result

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Objective:

• to identify carbohydrate from other biomolecules

Principle of Molisch’s test:

Molisch’s test is a general test for all carbohydrates. In this test, carbohydrates when reacted with conc. H2SO4 get dehydrated to form furfural and its derivatives.

When monosaccharide are treated with conc H2SO4 or conc HCl, -OH group of sugar are removed in the form of water and furfural is formed from pentose sugar and hydroxymethyl furfural is formed from hexose sugar. These products reacts with sulphonated α- naphthol to give a purple (violet red) colored complex.

Reagents for Molisch’s test:

• test solution: 5 % Glucose, 5 % Sucrose, 5 % Starch
• Molisch’s reagent (5 % α naphthol in ethanol)
• H2SO4
• Dry test tubes
• pipettes

Procedure of Molisch’s test:

1. Take 2ml of sample in dry test tube.
2. Take 2ml of distilled water in another tube as control.
3. Add 2-3 drops of Molisch’s reagent to the solution.
4. Gently pipette 1ml conc. H2SO4 along the side of the tube so that two distinct layers are formed.
5. Observe color change at the junction of two layers.
6. Appearance of purple color indicates the presence of carbohydrates.

Result Interpretation of Molisch’s test:

Positive Molisch’s test: purple color complex

Negative Molisch’s test: no purple color

Molisch’s Test: Objectives, Principle, Reagents, Procedure and Result

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Objective:

• to detect reducing sugar in a given solution

Principle of Fehling’s test:

Fehling’s test is one of the sensitive test for detection of reducing sugars. Fehling’s reagents comprises of two solution Fehling’s solution A and solution B. Fehling’s solution A is aqueous copper sulphate and Fehling’s solution B is alkaline sodium potassium tartarate ( Rochelle salt). Rochelle salts (sodium potassium tartarate) present in the reagent acts as the chelating agent in this reaction.These two solution are mixed in equal amount before test.

On heating an aldehyde or reducing sugar with Fehling’s solution give reddish brown prepitate. Formation of red precipitate of cuprous oxide denotes the presence of reducing sugar.

Reagents:

• test solution: 5 % Glucose, 5 % Sucrose, 5 % fructose, 5 % Lactose, 5 % Starch
• Fehling’s reagent (solution A: CuSO4.5H2O
• Fehling’s reagent ( solution B: Sodium potassium tartrate)
• Water bath
• Pipettes
• Dry test tubes

Procedure of Fehling’s test:

1. Take 1ml of sample in dry test tube.
2. Take 1ml of distilled water in another tube as control.
3. Add 1ml of Fehling’s reagent (A and B) to all the tubes.
4. Keep in boiling water bath.
5. Look for the development of red precipitate.

Result interpretation:

• Positive Fehling’s test: reddish brown ppt ( glucose, fructose, lactose)
• Negative Fehling’s test: No red ppt (sucrose, starch)

Fehling’s  Test: Objective, Principle, Reagents, Procedure and Result

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Objective:

• to detect amino acid containing phenol group (hydroxyl group attached to benzene ring) ie. Tyrosine

Principle of Millon’s test:

Compounds containing hydroxybenzene radical react with Millon’s reagent to form red complexes. The only amino acid having hydroxybenzene ring is tyrosine. Thus, this test is specific for the amino acid tyrosine and the protein containing this amino acid. Tyrosine when reacted with acidified mercuric sulphate solution gives yellow precipitate of mercury-amino acid complex. On addition of sodioum nitrate solution and heating, the yellow complex of mercury-amino acid complex converts to mercury phenolate which is in red color.

Reagents:

• test solution: 1 % arginine, 1 % tyrosine, phenol solution
• Millon’s reagent (Acidified mercuric sulphate)
• 1 % sodium nitrite

Procedure of Millon’s test:

1. Take 1ml test solution in dry test tube.
2. Similarly, take 1ml distilled water in another test tube as control.
3. Add 1ml of Millon’s reagent and mix well.
4. Boil gently for 1 minute.
5. Cool under tap water.
6. Now add 5 drops of 1 % sodium nitrite.
7. Heat the solution slightly.
8. Look for the development of brick red precipitate.

Result interpretation:

• Positive Millon’s test: Brick red color (Tyrosine and phenol solution)
• Negative Millon’s test: no red color ( arginine)

**all phenol give positive Millon’s test

Millon’s test: Objective, Principle, Reagents, Procedure and Result

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Objective:

• to detect amino acid gauanidium group [R-NH-C= (NH2)2+-NH2]. i.e arginine

Principle of Sakaguchi test:

This test is specific for arginine because this reaction is given by guanidinium compound. The arginine reacts with α – napththol and an oxidizing agent  such as bromine water or sodium hypochlorite/sodium hypobromite to give a red colored product. The other guanidinium containing compounds other than amino acid also give this reaction.

Reagents:

• test solution: 1 % arginine, 1 % glycine, 5 % egg white (albumin)
• 1 % α naphthol in alcohol
• Sodium hypochlorite
• 1 % urea solution
• Dry test tubes
• Pipettes

Procedure of Sakaguchi test

1. Take 1ml test solution in dry test tube.
2. Similarly, take 1ml distilled water in another test tube as control.
3. Add 2 drops of α naphthol and mix well.
4. Now add 2ml sodium hypochlorite to all test tubes.
5. Immediately add 1ml of urea solution to establish the red complex formed.

Result interpretation:

• Positive sakaguchi test: Red color ( Arginine)
• Negative sakaguchi test: no red color ( glycine, albumin)

Sakaguchi test: Objective, Principle, Reagents, Procedure and Result

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