Hemoglobin determination.pdf

HEMOGLOBIN DETERMINATION ( CYANMETHEMOGLOBIN METHOD )  Introduction ◼ The hemoglobin molecule is made up of heme and globin. ◼ Heme : is a pigment which contain iron. and its make up about 4% of the hemoglobin molecule. ◼ Globin: is a colorless protein. It makes up...

HEMOGLOBIN DETERMINATION ( CYANMETHEMOGLOBIN METHOD )  Introduction ◼ The hemoglobin molecule is made up of heme and globin. ◼ Heme : is a pigment which contain iron. and its make up about 4% of the hemoglobin molecule. ◼ Globin: is a colorless protein. It makes up about 96% of the hemoglobin molecule.  Introduction ◼ Hemoglobin is often abbreviated Hb. Or Hgb. ◼ Hemoglobin’s main function in the body is to carry oxygen from the lung to the tissues & to assist in transporting carbon dioxide from the tissues to the lungs.  Introduction ◼ The formation of Hgb takes place in the developing red cells located in bone marrow. ◼ Hgb in circulating blood is a mixture of hemoglobin,oxyhemoglobin,carboxyhemo- globin and minor amounts of other forms.  Introduction ◼ Each gram of Hgb can carry 1.34 ml of oxygen. ◼ The oxygen-combining ability of the blood is in direct proportion to the Hgb concentration, rather than the numbers of RBC’s ,because some cells contain more hemoglobin than others.  Principle of Hemoglobin Determination ◼ Blood is mixed with reagent solution called Drabkin’s solution which contains ferricyanide and cyanide. ◼ The ferricyanide oxidizes the iron (ferrous ions)(Fe2+) in the hemoglobin to the ferric (Fe3+) state , thereby changing hemoglobin to methemoglobin.  Principle of Hemoglobin Determination ◼ Methemoglobin subsequently reacts with the cyanide ions provided by potassium cyanide to form cyanmethemoglobin (HiCN). ◼ The cyanmethemoglobin produces a color ( brown color )which is measured in a colorimeter or spectrophotometer. ◼ In hemoglobin determination experiment, we use Spectrophotometry method to Measure how much a Cyanmethemoglobin absorbs light by measuring the intensity of light as a beam of light passes through sample solution.  Principle of Hemoglobin Determination K+ Ferricyanide Hemoglobin (Fe+²) Methemoglobin (Fe+³) Oxidation + K+ Cyanide Cyanmethemoglobin (HiCN)  Normal values ◼ Adult males : 14 – 18 gm/dl. ◼ Adult females : 12 – 16 gm/dl. ◼ Newborn : 17 – 19 gm/dl.  Materials 1. Alcohol wipes. 2. Hb pipettes or 20 ul micropipette. 3. Cotton. 4. Tapes. 5. Colorimeter or spectrophotometer. 6. Test tubes (cuvett).  Materials 7. Drabkin’s solution (yellow). Consist of : A. Potassium ferricyanide 200 mg. B. Potassium cyanide 50 mg. C. Potassium dihydroen phosphate 140 mg. D. Triton-X 1ml. E. Distilled water to 1L. 8. Hemoglobin standard curve  Procedure 1. Add 5 ml of Drabkin’s reagent to a test tube. 2. Add 20ul blood to the test tube using Hb pipette or micropipette. 3. Mix blood with Drabkin’s and wait for 5 to10 minute for the reaction to take place. 4. Measure the absorbance of the sample against blank (Drabkin’s reagent) at 540 nm by spectrophotometer. 5. According to the absorbance,get the Hb concentration by using standard Hb curve. Hemoglobin standard curve  Clinical Significant of The Test ◼ Hemoglobin determination is used to screen for anemia, to identify the severity of anemia, and to assist in evaluating the patient’s response to anemia therapy. ◼ Hgb values may vary according to physiological or pathological conditions.  Clinical Significant of The Test A. Physiological variation : Physiologically ,Hgb values are affected by age , sex , pregnancy , and altitude. Newborn infants have higher Hgb values than adults. Men have higher Hgb values than women. Clinical Significant of The Test Changes in altitude affect Hgb values,at a high altitude , there is less oxygen in the air , resulting in an increase in RBC’s count and Hgb values. On contrast , at a low altitude there is more oxygen in the air, resulting in a decrease in RBC’s count and Hgb values. Clinical Significant of The Test In pregnancy , the body gains fluid. Therefore , the RBC’s become less concentrated , consequently ,the RBC’s count falls. Since Hgb is contained in the RBC’s , the Hgb concentration also falls.  Clinical Significant of The Test B. Pathological variation : Decreased levels of hemoglobin : Because hemoglobin is a component of all RBC’s , the conditions that cause a low RBC’s , such as blood loss and bone marrow Suppression , also produce a low Hgb level.  Clinical Significant of The Test Hgb levels are lowered in patients who have abnormal types of Hgb (hemoglobinopathies). Some patients have normal RBC’s count but a low Hgb level. this situation occurs with iron deficiency anemia ,in which RBC’s have less Hgb than normal. Hgb values drop below normal in anemia and Leukemia.  Clinical Significant of The Test Increased levels of hemoglobin : Are found in any condition in which the # of circulating RBC’s rises above normal. Examples of conditions associated with increases in hemoglobin above the normal : -polycythemia vera. -in dehydration condition.  MECHANICAL SOURCES OF ERROR ◼ Pipetting error. ◼ Use of dirty or scratched cuvettes. ◼ Use of deteriorated reagent.  videos ◼ https://www.youtube.com/watch?v=kfB8B T98S34&ab_channel=swicmltvideos