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Lesson 6-3 Hemoglobin

GENERAL INFORMATION

Hemoglobin is a conjugated protein composed of the basic protein globin linked to 4 heme molecules. Ninety-eight percent of all the iron found in the blood is contained in hemoglobin. Hemoglobin transports oxygen and carbon dioxide. This important substance reacts with oxygen to form oxyhemoglobin. In the tissues, oxygen is released and reduced hemoglobin is formed. Hemoglobin can react with acids, bases, and oxidizing and reducing agents. It also can exist in a variety of forms. These hemoglobin compounds and variants are discussed briefly in the following paragraphs. For more detailed information, refer to the standard hematological texts.

COMPOUNDS OF HEMOGLOBIN

Oxyhemoglobin. Oxygen combines loosely with iron (ferrous state) in hemoglobin. The loosely attached oxygen diffuses into the tissues for oxidative processes. The hemoglobin then binds carbon dioxide and exists as reduced hemoglobin.

Carboxyhemoglobin. Hemoglobin combines with carbon monoxide to form carboxyhemoglobin. Carbon monoxide has an affinity 200 times greater for hemoglobin than oxygen does. Hemoglobin in this combination is incapable of oxygen transport.

Methamoglobin. This compound is formed when the ferrous state of the heme is oxidized to the ferric state. This compound is incapable of oxygen transport.

Sulfhemoglobin. This compound results from the combination of inorganic sulfides and hemoglobin. This compound is incapable of oxygen transport. This is an irreversible reaction.

Cyanmethemoglobin. This compound results when methemoglobin combines with the cyanide radical. This compound is used in hemoglobinometry.

VARIATIONS OF HEMOGLOBIN

The variations of hemoglobin occur due to structural differences in the globin protein. These differences are genetically controlled. The normal hemoglobin components are hemoglobin A (HbA), hemoglobin A2 (HbA2), and fetal hemoglobin (HbF). HbA constitutes most of the hemoglobin of a normal adult while HbA2 constitutes a much smaller amount. HbF is present during the first 4 to 6 months of life and not normally present in adults. Hemoglobin S and hemoglobin C are the most commonly occurring abnormal hemoglobins. Others (D, E, H, etc.) are found in rare occurrences associated with several types of anemia. The various types of hemoglobin are separated by electrophoresis.

HEMOBLOGINOMETRY

The hemoglobin concentration is directly proportional to the oxygen-combining capacity of blood. Therefore, the measurement of the hemoglobin concentration in the blood is important as a screening test for diseases associated with anemia and for following the response of these diseases to treatment. There are four basic ways to measure the hemoglobin concentration:

(1) measurement of the oxygen-combining capacity of blood (gasometric)
(2) measurement of the iron content (chemical method)
(3) colorimetric measurement of specific gravity (gravimetric method)
(4) the cyanmethemoglobin method is the method of choice and is most widely used

The cyanmethemoglobin is recommended by the Technical Subcommittee on Hemoglobinometry of the International Committee for Standardization in Hematology.

CHYANMETHEMOGLOBIN METHOD

Principle. Blood is diluted with a dilute solution of potassium ferricyanide and potassium cyanide at a slightly alkaline pH. The ferricyanide converts the hemoglobin to methemoglobin. The cyanide then reacts with the methemoglobin to form the stable cyanmethemoglobin. The color intensity is measured in a spectrophotometer at a wavelength of 540 mm. The optical density is proportional to the concentration of hemoglobin.

Discussion.

Cyanmethemoglobin is the most stable of the various hemoglobin pigments showing no evidence of deterioration after 6 years of storage in a refrigerator. The availability of prepared standards is a distinct advantage of this technique. All hemoglobin derivatives are converted to cyanmethemoglobin with the exception of sulfhemoglobin.

This method is highly accurate and is the most direct analysis available for total hemin or hemoglobin iron. Its disadvantage is the use of cyanide compounds, which, if handled carefully, should present little hazard.

For accuracy in hemoglobin determinations, it is absolutely necessary that the spectrophotometer and Sahli pipets be accurately calibrated.

Venous samples give more constant values than capillary samples.

If the procedure is performed properly, the degree of accuracy is +2 to 3 percent.

Normal Values.

Infants at birth: 15 to 20 g/dL.
Males: 13 to 18 g/dL.
Females: 12 to 16 g/dL.

DETECTION OF HEMOGLOBIN S AND NON S SICKLING HEMOGLOBINS

Principle. Erythrocytes are introduced into a phosphate buffer solution containing a reducing agent and hytic agent. The red cells are lysed and the hemoglobin is reduced. Reduced sickling types of hemoglobin are insoluble in phosphate buffer and turbidity results. On addition of urea, hemoglobin S dissolves.

Reagents.

(1) Add the entire content of one vial of Sickledex reagent powder to one bottle of Sickledex test solution. (Reagents are available commercially.)
(2) Dissolve the Sickledex reagent powder completely in the Sickledex test solution by shaking the bottle vigorously for a few seconds. The Sickledex test solution is then ready for use. Date the reconstituted test solution. This reagent remains stable under refrigeration at 4șC for approximately 60 days. See figure 6-1.

Figure 6-1. Sickledex tube test interpretation.

Procedure

(1) Pipet 2 ml of Sickledex reagent in 12 x 75 mm test tube.
(2) Add 0.02 ml of well-mixed anticoagulated blood (collected in EDTA).
(3) Mix the contents and al1a.v to stand at room temperature for a minimum of 6 minutes (see figure 6-1).
(4) After 6 minutes examine the tube for turbidity against a lined reader. Hemoglobin five, if present, produces turbidity in the tube.

Sources of Error

(1) Other unstable hemoglobins.
(2) Out dated reagents. The freshness of this reagent must be checked with positive and negative controls.
(3) Hemoglobin concentration less than 7 g/dL can cause a false negative
(4) False negative results could occur if the blood sample for testing is drawn within 4 months of transfusion.

Discussion

(1) Hemoglobin S is an inherited type of hemoglobin found primarily in blacks and people from Mediterranean areas.
(2) The degree of erythrocyte sickling is dependent on the concentration of hemoglobin. SS, SC, and SD cells sickle more rapidly than AS cells. Newborns with sickle cell anemia have erythrocytes more resistant to sickling due to the presence of hemoglobin F.
(3) The dithionite test also detects other sickling types of hemoglobin. Urea causes hemoglobin S (and structural variants of hemoglobin S) to dissolve. Other hemoglobins remain turbid in the presence of urea.
(4) This test is a rapid screening test for hemoglobin S. All positive dithionite tests should be electrophoresed for confirmation.

Interpretation. Hemoglobin S causes turbidity in the tube. Hemoglobin A is soluble in the phosphate buffer.

HEMOGLOBIN ELECTROHORESIS (CELLULOSE ACETATE)

Principle. Hemoglobin fractions are separated by the rate of their protein migration in an electrical medium. The fractions are stained with ponceau S and quantitated on a densitometer. The order of mobility from the cathode toward the anode is A3> AI> F> S-D>C-A2.

Discussion.

(1) A2 hemoglobin migrates identically to hemoglobin C. They are distinguished by the quantity present. If this band is 40 percent or more of the total hemoglobin, it is C. A2 hemoglobin should always be less than 20 percent.
(2) Two slow-moving, nonhemoglobin components are seen using this technique. These fractions are carbonic anhydrases I and II (CAI and CAII)
(3) Hemoglobin A2 is elevated in thalassemia minor.
(4) Genotype SS is found in patients with sickle cell anemia.
(5) Genotype AS is found in patients with sickle cell trait.
(6) This method separates hemoglobin A2 in the presence of hemoglobin S in patients manifesting sickle-thalassemia disease.
(7) Hemoglobin F is quantitated by the alkali denaturation test because it migrates close to the hemoglobin A1 fraction on the electrophoretic pattern.
(8) Include known A, S, and C controls in each analysis.
Normal Values
(1) A3 Hemoglobin: One and three tenths to 3 percent.
(2) Genotype: AA.
(3) F Hemoglobin: 0 to 2 percent (except in infants).

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