Hemoglobin and Blood Substitutes

Hemoglobin is mainly released from hemolysis of RBC. Hemolysis can occur in vivo, during venipuncture and blood collection or during processing of the sample. Hemoglobin interference depends on its concentration in the sample. Serum appears hemolyzed when the hemoglobin concentration exceeds 20 mg/dL (10). However, icteric serum may contain higher concentration of hemoglobin before hemolysis can be noticed. Hemoglobin interference is caused not only by the spectrophotometric properties of hemoglobin but also by its participation in chemical reaction with sample or reactant components as well (11). The absorbance maxima of the heme moiety in hemoglobin are at 540- to 580-nm wavelengths. However, hemoglobin begins to absorb around 340 nm, absorbance increasing at 400-430 nm as well. The iron atom in the center of the heme group is the source of such absorbances. Of the many variants of hemoglobin, methemoglobin (where the iron is in 3+ oxidation state) and cyanmethemoglobin (cyanide complex of hemoglobin) also absorb at 500 and 480 nm, respectively. Methods that use the absorbance properties of NADH or NADH (340 nm) may thus be affected by hemolysis. When hemoglobin is oxidized to methemoglobin, the absorbance at 340 nm decreases.

In renal failure as well as damage to kidney or urinary pathways, the heme or its derivatives may be present in urine, generating hemolytic interference similar to that of serum. Urine may also have interference from myoglobin, the oxygen-binding protein in striated micelles, also containing the heme group. Whereas hemoglobin is a tetramer of the heme and globin complex, myoglobin is a monomer and, consequently, is smaller than hemoglobin by three-fourths. Thus, when there is injury to skeletal or cardiac muscle, myoglobin may be released and then excreted in urine (myoglobinuria). Under such condition, urine samples have a color similar to that of a cola drink or black coffee and show interference similar to that of hemolysis in serum.

If the type of blood for a patient is in short supply, many hospitals now use blood substitutes, which are mostly derivatized or polymerized hemoglobin. The blood substitutes interfere in many analyses in the same way as hemoglobin. Thus, it was demonstrated that Hemolink®, an O-raffinose cross-linked hemoglobin blood substitute, showed positive or negative interference in many routine chemistry and immunochem-istry assays (12). Another type of blood substitute is polyfluorocarbon, which also has been reported to interfere with immunoassays.

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