Hemoglobin Estimation

HAEMOGLOBIN ESTIMATION • Hemoglobin is the main component of red blood cells and it is conjugated protein • A molecule of Hb contains two pair of polypeptide chains and four haem groups • Iron content of Hb is 0.347 gm/100 g • The main function of Hb is transport oxygen from lungs to tissue • It is composed of heme (iron( ferrous) + protoporphyrin ) and globin polypeptide chain • Normal hemoglobin variants are • Embryonic hemoglobin (Gower II ,Gower I, Portland ) • • • Fetal hemoglobin (Hb F) Adult hemoglobin (Hb A2 ) (Hb A) Various forms of Hemoglobin are : ▪ ▪ Oxy hemoglobin (Hb O2) Carboxy hemoglobin (Hb CO ) ▪ Sulf hemoglobin (S Hb) ▪ Methaemoglobin (Hi ) INDICATIONS ❖ To determine the presence and severity of Anemia ❖ Screening for polycythemia METHODS Various methods are available for estimation of hemoglobin in the laboratory ❖ PHYSICAL METHOD ❖ CHEMICAL METHOD ❖ GASOMETRIC METHOD ❖ COLORIMETRIC METHOD ❖ PHYSICAL METHOD ➢ Based on the assumption that the changes in the specific gravity of blood is entirely due to changes in Hb concentration ➢ It is measured by using cuso4 solution of known specific gravity Basic principle : specific gravity of cuso4 =1.053 with the Hb equivalent to 12.5 gm CHEMICAL METHOD ◄ Accurate and reference method ◄ Iron content is estimated by wongs method ◄ Priniple : 1 gram of Hb =3.47 mg of iron ◄ In this method protein are first precipitated by tungstic acid , then iron is detached from the Hb by treating with sulphuric acid & potassium thiocynate GASOMETRIC METHOD ◄ Oxygen carrying capacity is measured by van slyke apparatus ◄ 1gm Hb bind about 1.34 ml of O2 ◄ It measure only functional Hb ◄ Does not measure carboxy hb ,meth hb ,sulf hb COLORIMETRIC METHOD ◄ Based on mesuring the colour of hb or any derivative in the blood sample Visual colorimetry ❖ DIRECT MATCHING TALLIQVIST METHOD SPENCERS METHOD DARES METHOD ❖ CARBOXY HEMOGLOBIN METHOD HALDANES METHOD ❖ ACID HAEMATIN METHOD SAHLIS METHOD HADEN HAUSSER METHOD NEW COMERS METHOD Photo electric colorimetry Alkaline hematin method Oxy hemoglobin method Cyan meth hemoglobin method SAHLIS /ACID HEMATIN METHOD Principle: Blood is mixed with N/10 HCl resulting in the conversion of Hb to acid hematin which is brown in color. The solution is diluted till it’s color matches with the brown colored glass of the comparator box. The concentration of Hb is read directly. CYAN METH HEMOGLOBIN METHOD This is the internationally recommended method for determining hemoglobin Principle: Blood is diluted in a solution containing potassium cyanide and potassium ferricyanide. The latter converts Hb to methemoglobin which is converted to cyanmethemoglobin (HiCN) by potassium cyanide. The absorbance of the solution is then measured in a spectrophotometer at a wavelength of 540nm or in a colorimeter using a yellow green filter OXYHEMOGLOBIN METHOD Hb is converted to oxyhemoglobin by reaction with ammonia and the color of the solution is measured in a photocolorimeter. The advantages of this method are that it is simple, quick The method does not give satisfactory results in the presence of carboxyhemoglobin, methemoglobin and sulphemoglobin. ALKALINE HEMATIN METHOD Blood is converted to alkaline hematin by addition of alkali such as sodium hydroxide and the color measured in a colorimeter at 540 nm. It gives a true estimate of Hb and is not affected by the presence of plasma proteins and lipids. However, it is not used routinely as it is less accurate than the cyanmethemoglobin method and some forms of Hb such as HbF are resistant to alkali denaturation. Significance of Hb estimation: Hemoglobin estimation is used as a screening test for detecting anemia. Selection of blood donars Screening of polycythemia WHO REFERENCE RANGE : males : 13-17 gm/dl Female : 12 – 15gm/dl PREVIOUS MCQ Drabkins fluid contains the following except …….? a) Potassium cyanide b) potassium ferricyanide c) sodium dihydrogen phosphate d) sterose se Which of the following transport Hb ? a) Haptoglobin b) Hemopexin c) Transferrin d) Albumin The major breakdown product of Hb is ? a) Protein b) Iron c) Bilirubin d) glucose