Mechanism of interference by hemolysis in immunoassays and requirements for sample quality.

To the Editor: Cardiac troponin I (cTnI) is considered organ-specific, and few circumstances other than heart muscle injury produce false increases in plasma. We encountered unexpectedly high cTnI in a two-site, enzyme-linked immunoassay using poly-clonal goat antibody (1) when by mistake, two visually clear but grossly hemolyzed samples from one patient were not rejected by the laboratory. Hemoglobin (Hb) reportedly is not an interferent in the test: ". .. levels of the following do not appear to interfere.. . hemoglobin 2,000 mg/ dL " (2). Elsewhere, the manufacturer states that: " Plasma samples should be clear to straw in color " (3) and ". .. strongly hemolyzed.. . samples should be avoided " (4). No data are presented that hemolysis interferes. These statements suggested that " he-molysis " , rather than " hemoglobin " , interfered. Off-label tests were carried out in which known volumes (250, 500, and 1000 ␮L) of three patient plasma samples were mixed with known volumes (5, 10, 20, 40, 60, 80, and 320 ␮L) of erythrocytes from the same patient. The plasma samples had produced cTnI data below the limit of detection (Ͻ0.5 ␮g/L) before mixing. The mixtures were frozen and thawed once to lyse cells. According to the manufacturer, one freeze-thaw cycle does not alter pure plasma cTnI data. Analysis of a control specimen, in which freeze-thawed plasma was added to a plasma sample that had a measured cTnI concentration of Ͻ0.5 ␮g/L before freezing, showed Ͻ0.5 ␮g/L as well. Estimates of hemolysis were made by determining a hemolysis index (HI) for each sample using a Hitachi 911 instrument: " HI is reported in hemolysis units corresponding to he-moglobin concentration in mg/dL. These units are linear and semiquan-titative. A hemolysis index of 500 is equivalent to a known hemoglobin concentration of approximately 500 mg/dL " (5). When hemolysate was added to plasma, cTnI was increased from reference values to high values in a dose-related way (Table 1). The man-ufacturer's interference study was performed by adding high concentration Hb stock into samples. This confirmed that the addition of human methemoglobin (Sigma Diag-nostics, cat. no. 525-18) in concentrations of up to 30 g/L (3000 mg/dL) did not affect cTnI results. Recentrifu-gation (10 min at 2000g) of hemolyzed specimens that were adversely affected eliminated interference in 18 samples , but not in 2 (which produced cTnI values of 3.9 and 11.2 ␮g/L). Our results raise several points …