How to estimate Hb concentration ?

Hemoglobin is the main intracellular conjugated protein present in the red blood cells which gives red color to the blood. It is made up of large protein molecule globulin and four ring like structure each containing one atom of iron. Its main function is to transport oxygen to the tissues and to remove the carbon dioxide from the tissues by the lungs.

METHOD OF ESTIMATION

1. Colorimetric Method

Ø  Tallquist Method

Ø  Sahli's Acid Hematin Method

Ø  Haldane Method

Ø  Dare Method

Ø  Spencer Method

Ø  Photoelectric Method

§  Oxyhemoglobin Method

§  Cyanmethemoglobin Method

2. Specific Gravity Method (Physical Method)

3. Iron Content Method (Chemical Method)

4. Oxygen Combining Capacity Method (Gasometric Method)

MANUAL METHOD

Sahli's Acid Hematin Method

Principle

Hemoglobin is converted to acid hematin by the addition of N/10 or 0.1N hydrochloric acid. It gives the brown color, which is compared with standard glass reference blocks. The intensity of brown color depends upon the amount of acid hematin, which is directly proportional to the concentration of hemoglobin present in the specimen.

Requirements

1.Sahli's Hemoglobinometer

       It consists of

• Standard brown glass mounted on a comparator.
• Graduated tube
• Hb pipette.
• Stirrer

2. 0.1N HCl and dropper

3. Distilled water

4. Pasteur pipette.

Procedure

Ø  By using a Pasteur pipette, add 0.1 N HCl in the tube up to the lowest mark.(20% mark)

Ø  Draw blood up to 20μL mark in the pipette.

Ø  Adjust the blood column, carefully without bubbles.

Ø  Wipe excess of the blood on the sides of the pipettes by using a day piece of cotton.

Ø  Transfer blood to the acid in the graduated tube.

Ø  Rinse the pipette well mix well the reaction mixture and allow the tube to stand at least 10 minutes.

Ø  Dilute the solution with distilled water by hemoglobin estimation.

Ø  By adding few drops at a time carefully and by mixing the reaction mixture until the color matches with the glass plate in the comparator.

Ø  The matching should be done only against natural light and the reading of the fluid is noted at its lower meniscus and the reading corresponding to this level on the scale is recorded in gm/dl.

Note

Precautions

·         Avoid squeezing the finger tip while drawing blood.

·         Avoid air bubbles in the pipette.

·         Wipe the tip of pipette before transferring blood into the hemoglobinometer tube.

·         Blow the blood directly into the HCl into the hemoglobinometer tube quickly and rinse it with the HCl

·         While matching the color:

 §  Hold the hemoglobinometer against bright light
§  Graduations on the tube should not be visible.
§  Don’t block the passage of light through tube with the fingers.
§  Raise the stirrer above the fluid level while matching the color.
§  Don’t remove the stirrer out of the tube once it is introduced into it.

Cynmethhemoglobin method

Principle

Drabkin's reagents contain potassium ferricyanide and potassium or sodium cyanide. When whole blood is mixed with Drabkin's reagent, the hemoglobin present in whole blood first converted to the methemoglobin (unstable) by the action of potassium ferricyanide and this methemoglobin is further converted to stable cyanmethemoglobin by the action of potassium or sodium cyanide. The resultant color complex formed is directly proportional to the hemoglobin concentration, which is directly measured spectrophotometrically at 540nm.

Requirements

1. Cyanmethemoglobin solution (Drabkin's reagent)

Sodium Bicarbonate	1gm
Potassium Ferricyanide	200mg
Potassium Cyanide	50mg
Nonionic Detergent	0.5ml

 Make up to 1000ml.with distilled water. Store in brown color bottle in refrigerator.

2. Test tubes (15 x 12.5mm)

3. Calorimeter

4. Hemoglobin pipette

5. Standard of cyanmethemoglobin

Procedure

Ø  Pipette 5 ml of cyanmethemoglobin reagent in test and blank.

Ø  Add 0.02ml of whole blood in test.

Ø  Wait for 5 mins.

Ø  Read the absorbance of blank

Ø  Read the test value at 540 nm.

Ø  Read the absorbance of standard (15gm/dl) by pipettes it directly in a cuvette

Calculation

 Hb concentration = OD of test/OD of standard x 15

v  In the blood bank during the screening the test of donor the specific gravity hemoglobin method is done for the hemoglobin estimation by using coppersulphate solution.

Principle

The hemoglobin concentration of the given sample depends upon its specific gravity which is calculated by using copper sulphate solution.

Procedure

Ø  A drop of blood is allowed to fall into CuSO4 solution of varying specific gravity

     §  If the drop of blood sinks specific gravity of blood is greater than the solution
   §  If the drop of blood floats specific gravity of the blood is lesser than the solution

Ø  It does not exact estimate the concentration. It only determines whether the person’s hemoglobin is above the specified level or not. In blood bank a solution of specific gravity 1.047 is used and is equivalent to the hemoglobin level of 10.5gm%

Normal value

Men                    15.5±2.5gm%

Women               14±2.5gm%

New born            16.5±3gm%

Children              12-13gm%

Clinical Significance

A. Increased

·         In polycythemia

·         After vigorous exercise

·         Excitement

·         Hemoconcentration like dehydration, burns, severe vomiting, and intestinal obstruction

·         Congenital heart disease

B. Decreased

·         In anemia

·         Drugs that cause aplastic anemia then cause hemolysis

·         Pregnancy

·         Premature infants

Note

Ø  Protein, lipid bilirubin, methemoglobin, carboxyhemoglobin, sulfhemoglobin, influence the depth of the color and cannot be converted to acid hematin by HCL.

Ø  Cyanmethemoglobin estimate all the hemoglobin except sulfhemoglobin.

Ø  In Drabkin's solution instead of sodium bicarbonate potassium dihydrogen phosphate buffer can also be used which shortens the time needed for complete conversion of hemoglobin to cyanmethemoglobin .The non ionic detergent enhances the lysis of erythrocytes and decreases turbidity from protein precipitation.

Ø  Since the potassium cyanide present in Drabkin’s solution is poisonous it should be disposed in running tap water.

Ø  The mean hemoglobin in the dark persons is lower than in whitish.

Ø  Falsely high results may occur due to prolonged venous stasis during venipuncture

    

The rough value for Hb  estimation can be done by

PCV/3

   Or

RBC count x 3

Introduction: Pathology Lab

Introduction to Pathology

Pathology is the literally the study (logos) of suffering (pathos). More specifically, it is a bridge that involves basic science and a clinical practice and is devoted to the study of structural and functional changes in cells, tissues, and organs that underlie the disease. By the use of molecular, microbiological, immunological and morphological techniques, pathology attempts to explain the whys and wherefores of the signs and symptoms manifested by the patient while providing a sound foundation for rational clinical care or a therapy. Clinical pathology reflects major current use of laboratory measurements  and examination in patients care management as well as diagnostic application.

The main purposes of laboratory clinical pathology are:-

Ø  Confirm or reject the diagnosis

Ø  Provide the guidelines in patients managements including test utilization properties

Ø  Establish a prognosis

Ø  Detect disease through case finding or screening

Ø  Monitoring follow up therapy.

Clinical pathology has 3 branches under that and they are:

A. Cytology or Clinical Hematology

B. Immunohematology

C. Histology