Hemoglobin Determination

Hemoglobin

• Hemoglobin— Found in the RBC.— Responsible for carrying oxygen to all cells in the body.— Also binds to carbon dioxide and carries it to the lungs from the cells to be released.

• An accurate measurement of hemoglobin in the body gives the health care worker an idea of the patient’s oxygen carrying capacity.

• Hemoglobin detection can also give the health care worker of current blood loss and recovery from blood loss.

• Monitoring hemoglobin can also aid in the treatment of RBC disorders, like anemia.

Hemoglobin

• Hemoglobin— Makes up 98% of the protein found in the RBC.— Gives blood its red color.— Composed of two parts….1. Heme 4 iron atoms in the ferrous state (Fe2+)2. Globin 4 protein chains (genetically determined) The most common globin forms are alpha and beta chains. Hemoglobin's are named based on the structure of the protein chain that is present.• Hemoglobin A 95-98% of adult hemoglobin• Hemoglobin A2 3-5% of adult hemoglobin

• Hemoglobin F normal fetal hemoglobin, <1% of adult hemoglobin• Hemoglobin S abnormal hemoglobin found in sickle cell anemia

Hemoglobin

• Each hemoglobin molecule contains four heme groups (iron molecules), each associated with its own protein chain.

• Iron is a must for hemoglobin synthesis.

• Iron is reused by the body as needed.

• When old RBCs die the iron is recycled to make new hemoglobin molecules.

• If the body doesn’t have enough iron, hemoglobin production decreases and the RBCs will not have enough hemoglobin.

• This can result in anemia causing patients to experience fatigue and paleness of skin.

• Excessive blood loss can also lead to low iron levels and poor hemoglobin production.

Reference Values for Hemoglobin

• Reference values for hemoglobin depend on gender and age.• Hemoglobin is higher at birth and lowers with childhood and increases again

as you get older.• At birth hemoglobin levels range from 16-23 g/dL• During childhood hemoglobin levels range from 10-14 g/dL• Adult males have hemoglobin levels ranging from 13-17 g/dL• Adult females have hemoglobin levels ranging from 12-16 g/dL• Your hemoglobin level should be 1/3 of your hematocrit level.• Exercise as well as diet affect the level of hemoglobin you have in your

system.• If you exercise a lot your hemoglobin will be higher because you need more

oxygen carrying capacity.• If you have a diet low in iron your hemoglobin level may be lower than

normal.

Reference Values for Hemoglobin

• Determining hemoglobin value

— There are several ways to determine a person’s hemoglobin concentration.

— Methods include….. Specific Gravity TechniqueCyanmethemoglobinAzidemethemoglobin

—Some methods can be performed manually while others are performed on a hematology or POCT analyzer.

Reference Values for Hemoglobin

• Determining hemoglobin value— Specific Gravity Technique Estimates the hemoglobin concentration.

Drop of blood is placed into a cupper sulfate solution.

The specific gravity of the solution must be from 1.052 to 1.054.

This is the same specific gravity of blood with a hemoglobin concentration at the low end of the reference range.

If the drop falls to the bottom quickly the specific gravity is higher than the solution and the hemoglobin is in the normal range.

If the drop floats or falls slowly the specific gravity is lower than the solution and the hemoglobin is below the normal range.

Common method in blood donation centers like the American Red Cross.

Reference Values for Hemoglobin

• Determining hemoglobin value

—Cyanmethemoglobin Blood is mixed with a reagent (Drabkin’s reagent) containing iron, potassium, cyanide, and sodium bicarbonate.

The Drabkin’s reagent and the hemoglobin in the blood combin to form a colored end- product known as cyanmethemoglobin.

A hematology analyzer or hemoglobinometer measures the end product photometrically.

Reference Values for Hemoglobin

• Determining hemoglobin value

— Azidemethemoglobin The azidemethemolgobin reagent contains a lysing chemical, an oxidizing chemical and azide.

The oxyhemoglobin which is in the form of ferrous iron is oxidized to ferric iron to form methemoglobin. The methemoglobin combines with azide to form azidemethemoglobin, that can also be measured photometrically by a hematology analyzer.

Reference Values for Hemoglobin

• Determining hemoglobin value

— POCT analyzers or hematology analyzers use the before mentioned methods to determine one’s hemoglobin.

— POCT analyzers require only a small drop or two of blood to determine the sample’s hemoglobin concentration.

— Hematology analyzers require more blood and test for more parameters than the hemoglobin alone and often times run an entire CBC.