biochemical tests. Statistical analysis of the results showed that hemolysis had the greatest effect on the lactate dehydrogenase, acid phosphatase, and potas-.
CLIN. CHEM. 38/4, 575-677
(1992)
Effect of In Vitro Hemolysis on 25 Common BiochemicalTests Dogan Y#{252}cel and Kiara Dalva Clinical chemists frequently encounter hemolyzed samples. Our study examines the effects of hemolysison the results of 25 common biochemical tests. We collected 60
15-mL blood samples from inpatients and outpatients and mechanically hemolyzed 10 mL of the samples in a two-stepprocedure.We classified serum from these samplesas beingnonhemolyzed,moderatelyhemolyzed, or severely hemolyzed and then performed 25 common biochemical tests. Statistical analysis of the results showed that hemolysis had the greatest effect on the lactate dehydrogenase, acid phosphatase, and potassium tests. Additional Keyphrases: sample handllng nase - add phosphatase - potassium
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lactate dehydrogevanatlon, source of
Hemolysis is common in blood specimens, occurring when blood contacts foreign surfaces. The use of partly hemolyzed serum may be unavoidable, especially during cardiac surgery or cardiac bypass, when there is transfusion or extracorporeal circulation. Hemolysis can result from improper drawing and handling of specimens and can occur during centrifugation and separation procedures. Therefore, clinical chemists must know whether hemolysis affects the analyses ordered by clinicians. Many laboratories reject all hemolyzed specimens without considering whether this approach is justified. Hemolysis in serum is visible when the hemoglobin (Jib) concentration is >3.1 &mol/L (1,2). Hemolysis has little effect on constituents that are present at lower concentrations in erythrocytes than in plasma, but a marked effect may be observed for constituents that are present at a higher concentration in erythrocytes than in plasma. Hb may also interfere directly in the colorimetric determination of constituents (2-4). The effects of hemolysis were thoroughly studied by several investigators who simulated hemolysis by adding lysed erythrocytes to plasma pooled from only a few subjects (2-6). Here we investigate the effects of hemolysis by correlating the results of biochemical tests with concentrations of free Hb in specimens hemolyzed by mechanical trauma. We think that this type of correlation gives data that are more useful for evaluating whether tests are affected by hemolysis.
Materials and Methods To study the effect of in vitro hemolysis on whole blood, we obtained 60 15-mL blood samples from pa-
tients in the coronary care, intensive care, and hemodialysis units of our hospital. With this distribution of patients, we hoped to have results over the entire range for each analyte tested. We drew the samples through wide-bore needles (18 gauge) with minimal stasis into glass tubes. We centrifuged 5 mL of each sample within 30 mm of collection, at 1500 x g for 10 mm, thus obtaining 2.0-mL samples of nonhemolyzed serum. We hemolyzed the remaining 10 mL of each sample by stirring with a metallic bar, 5 mL for 1 mm and 5 mL for 2 mm (mechanical trauma), and we obtained 2.0-mL samples of moderately and severely hemolyzed serum after centrifuging as before. This method of cell lysis was chosen because it is analogous to the mechanical disruption of erythrocytes that frequently occurs during faulty blood collection and sample preparation. Because the concentration of free Hb in serum is used as a measure of hemolysis, we measured free Hb in all specimens spectrophotometrically (7) and grouped the samples as nonhemolyzed (group I), moderately hemolyzed (group II), and severely hemolyzed (group ifi) (n = 60 each). We used a Dacos analyzer with Dart reagents (all from Coulter Electronics, Inc., Hialeah, FL) reagents to measure aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), urea, creatinine, cholesterol, high-density lipoprotein cholesterol (after precipitation with magnesium phosphotungstate), triglyceride, total and direct bilirubin, sodium, and potassium. We used an Encore analyzer (Baker Instruments Co., Allentown, PA) to measure glucose (glucose oxidase/peroxidase/phenol/ammnoantipyrine chromogenic system; equilibrium), albumin (dye binding with bromcresol green; kinetic), uric acid (uricase/peroxidase/phenol/ammoantipyrine chromogenic system; equilibrium), alkaline phosphatase (EC 3.1.3.1; 4-nitrophenyl phosphate as substrate), and lactate dehydrogenase (EC 1.1.1.27; pyruvate-+lactate reaction; ultraviolet, kinetic). We used a Vitatron SPS spectrophotometer (Vital Scientific, Dieren, The Netherlands) to measure calcium (cresolphthalemn complexone; equilibrium), magnesium (with Titan yellow after deproteinization; equilibrium), inorganic phosphate (with acid molybdate after deproteiiuzation; equilibrium), amylase (EC 3.2.1.1; amyloclastic method), lipase (EC 3.1.1.3; olive oil as substrate; turbidimetric), acid pho8phatase (EC 3.1.3.2; 4-nitrophenyl phosphate as substrate) and its prostatic isoenzyme (with tartrate inhibition), and Hb. We measured chloride with mercurimetric titration. Results
Clinical Biochemistry Laboratoiy, YOkaek Ihtisas Hastanesi (High Specialization Hospital), Sihhiye, Ankara 06100, Thrkey. Received September 9, 1991; accepted February 10, 1992.
The mean (and SD) free Jib concentrations for groups 1,11,and ifi were 0.744(0.496), 6.743(2.155), and 17.608 CLINICAL CHEMISTRY,
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(4.247) pmol/L, respectively. Differences of the means between groups I and II and groups II and ifi were statistically significant (P
