Abnormal Hemoglobin Testing PDF

In 1972, the Georgia General Assembly passed two laws concerning sickle cell testing. One requires testing for infants of the susceptible ethnic background and the other requires that sickle cell testing be offered to applicants for a marriage license. The intent of the laws is for the patient to be...

In 1972, the Georgia General Assembly passed two laws concerning sickle cell testing. One requires testing for infants of the susceptible ethnic background and the other requires that sickle cell testing be offered to applicants for a marriage license. The intent of the laws is for the patient to be aware that sickle cell disease is caused by an inherited abnormal gene. Since the sickle cell gene may interact with other abnormal hemoglobin genes to produce an anemia, screening is carried out via cellulose acetate electrophoretic separation at an alkaline pH. The principle of electrophoretic separation is fairly simple. Depending upon the alkalinity or acidity of the surrounding medium, hemoglobin molecules will carry either a tiny positive or negative electrical charge. If a hemoglobin molecule is in an acid buffer solution, the molecules will have a positive charge. When an electric current is applied, the molecules will move toward the negative electrode (called the cathode). If a hemoglobin molecule is in an alkaline buffer, the charge will be negative and movement will then proceed toward the positive electrode (called the anode). Each type of hemoglobin is different in its molecular structure and moves at different speeds. Samples are first centrifuged and a measured amount of the packed red blood cells are mixed with a solution that ruptures the cell membrane, freeing the hemoglobin. The cellulose acetate can be of varying sizes and holds up to 8 samples, one being a control consisting of a mixture of the common hemoglobin types: adult (A); fetal (F); sickle cell (S); and hemoglobin C (a rare disorder that causes an anemia). The samples are simultaneously applied to the membrane, which is then placed in the electrophoretic chamber and electric current is applied. The separation takes from 15-45 minutes depending on the equipment used. You can watch the reddish-brown samples separate into distinct bands. The fastest moving hemoglobin type is A, followed by F, then S, and finally C. Origin C S F A Adult Hgb (AA) Fetal Hgb (FF) Sickle Hgb (SS) C disease (CC) Cathode - Anode + Movement of hemoglobin molecules at pH 8.4, 450 voltage The genes an individual has for manufacturing hemoglobin are inherited from their parents, one gene from each parent. Most of us are AA: one A gene from each parent. A person with sickle cell trait inherits one A gene from one parent and one S gene from the other parent. When sickle cell disease occurs, the person inherits 2 S genes, one from each parent. There are less common hemoglobins such as D, E, and C. Each of these could be inherited along with S to produce an anemia, or in combination with one another. For example, one could get E hemoglobin from one parent and S hemoglobin from the other and have SE disease. This is called an interaction. Fetal hemoglobin is manufactured by infants, but by 6 months of age they are normally making adult hemoglobin. If fetal hemoglobin is noted in older persons, there may be a serious underlying cause, since it is also produced during many anemic conditions. However, fetal hemoglobin is also produced by relatively few normal individuals. From our preceding diagram you can see that each hemoglobin type separates into distinct bands, but this is not the entire story. Some types of hemoglobin molecules move at the same speed. For example, both S and D migrate at the same rate, as do C and E. There is a second test to differentiate S and D and C and E. Many times, specimens are sent to CDC to make positive identification of the presence of other types of hemoglobin. When a patient appears to have S or C hemoglobin we perform a solubility test. In the reduced state, meaning there is no oxygen, S hemoglobin and the very rare C ~Harlem~ hemoglobin, are highly insoluble in a concentrated phosphate buffer. This insolubility is a positive result for the solubility test. All other hemoglobins will return a negative solubility test result, including hemoglobin D.