Hemoglobin Concentration Determination

Hemopoiesis Is the process of blood cell formation which takes place during the embryonic life in the yolk sac; mesenchyme and blood vessels; liver; spleen, thymus and lymph nodes; bone marrow, while in late fetus & adult takes place in bone marrow and lymphtic tissues in normal situation (medullary hemopoiesis). In pathological conditions hemopoiesis is (extramedullary) in the liver, spleen and lymph nodes.

Hemoglobin (Hb) Hemoglobin (Hb) is the standard abbreviation for hemoglobin, the oxygen-carrying pigment and predominant protein in the red blood cells. Hemoglobin is the protein that carries oxygen from the lungs to the tissues and carries carbon dioxide from the tissues back to the lungs. In order to function most efficiently, hemoglobin needs to bind to oxygen tightly in the oxygen-rich atmosphere of the lungs and be able to release oxygen rapidly in the relatively oxygen-poor environment of the tissues. It does this in a most elegant and intricately coordinated way. Hemoglobin forms an unstable, reversible bond with oxygen. In its oxygenated state it is called oxyhemoglobin and is bright red. In the reduced state it is called deoxyhemoglobin and is purple-blue.

Structure of hemoglobin A hemoglobin molecule consists of four polypeptide chains: two alpha chains, each with 141 amino acids and two beta chains, each with 146 amino acids. The protein portion of each of these chains is called "globin". The α and β globin chains are very similar in structure and each one of them is liked with a heme molecule.

Structure of hemoglobin

Heme molecules A heme group is a flat ring molecule containing carbon, nitrogen and hydrogen atoms, with a single Fe2+ ion at the center. Without the iron, the ring is called a porphyrin. Changes in the amino acid sequence of these chains results in abnormal hemoglobin's. For example, hemoglobin S is found in sickle-cell disease, a severe type of anemia in which the red cells become sickle-shaped when oxygen is in short supply.

Hemoglobin facts and stats Each RBC contains 280 million Hb molecules. The average adult contains about 5 million RBCs /mm3. The average adult has about 5 litters of blood. Thus the average adult has about 790 gm of Hb.

Normal Human Hemoglobin's Adult hemoglobin's Fetal hemoglobin Embryonic hemoglobin's Hemoglobin A {2, 2} >95% hemoglobin A2 {2, 2} <3.5% hemoglobin F {2, 2} 1-2 % hemoglobin F gower gower Portland-

Normal Ranges In the very common laboratory test for hemoglobin (Hb), it is measured as total hemoglobin and the result is expressed as the amount of hemoglobin in grams (gm) per deciliter (dl) of whole blood, a deciliter being 100 milliliters. The normal ranges for hemoglobin depend on: The age. Altitude The sex of the person.

The normal ranges 17-22gmdl Newborns 17-22gm\dl One (1) week of age One (1) month of age 11-13gm\dl Children 14-18gm\dl Adult men 12-16gm\dl Adult women 12.4-14.9gm\dl Men after middle age 11.7-13.8gm\dl Women after middle age Normal values in an adult are 12 to 18 grams per deciliter (100 milliliters) of blood.

Above-normal hemoglobin levels is called polycythemia which is may be: Secondary polycythemia which is may be due to: Dehydration (sever burns, diarrhea, vomitting, …etc.). Severe lung or heart disease. Living at high altitudes. Heavy smoking. Primary polycythemia which is due malignant variation in blood cells production in bone marrow

Below-normal hemoglobin levels may lead to anemia that can be the result of: Iron deficiency or deficiencies in essential vitamins of other elements, such as B12, folate, B6. Inherited hemoglobin defects, such as sickle cell anemia or Thalassaemia. Other inherited defects affecting the red blood cells. Excessive bleeding. Excessive destruction of red blood cells. Kidney disease. Bone marrow failure or aplastic anemia. Cancers that affect the bone marrow.

Measurement of hemoglobin The Cyanmethemoglobin Method for Hb determination is the reference method. Principle: Whole blood is diluted in a solution of potassium Ferricyanide and potassium cyanide. The Hb is oxidize to methemoglobin by the potassium Ferricyanide. The potassium cyanide then converts the methemoglobin to cyanmethemoglobin. The absorbance of the cyanmethemoglobin at 540 nm is directly proportional to the Hb concentration. Sulfhemoglobin is not converted to cyanmethemoglobin; therefore, it can not be measured by this method.

Hb (Fe++) K3Fe (CN)6 methemoglobin (Fe+++ ) KCN Cyanmethemoglobin

Procedure of standard curve Create a standard curve, using a commercially available cyanmethemoglobin standard which, has constant concentration 25g/dl, the following dilutions should be made to get the line between the concentration & the absorbance of the standard using also drabkin reagent as shown:

Procedure V of Drabkin reagent / ml Volume of St / ml Absorbance reading Hb concentration g/dl 5 4 1 0.125 3 2 0.250 10 0.375 15 2.5 0.188 7.5

Procedure Transfer the dilutions to cuvettes. Starting with the blank, measure the absorbance on a spectrophotometer at 540 nm. Plot absorbance on the y-axis and the Hb concentration on the x-axis. The Hb concentrations of the patients’ samples and controls can be read from this standard curve.

Standard Curve 0.500 0.375 Absorbance 0.250 0.125 5 10 15 20 5 10 15 20 Concentration

Calculation Slope = abs (st) Con (st) 1 = Con (st) = Factor C (x) = Con (st) X abs (x) abs (st) C (x) = Factor X abs (x)