Interference of Blood Cell Lysis on Routine Coagulation Testing Giuseppe Lippi, MD; Martina Montagnana, MD; Gian Luca Salvagno, MD; Gian Cesare Guidi, MD c Context.ÐPreanalytical factors in¯uencing the reliability observed in activated partial thromboplastin time and ®- of laboratory testing are commonplace. It is traditionally brinogen in samples containing a ®nal lysate concentration accepted that hemolytic samples are unsuitable for coag- of 0.9%. The current analytical quality speci®cations for ulation assays because of the release of hemoglobin, intra- desirable bias are 62.0% for prothrombin time, 62.3% cellular components, and thromboplastic substances from for activated partial thromboplastin time, and 64.8% for damaged blood cells. ®brinogen. Percent variations from the baseline values ex- Objective.ÐTo evaluate the in¯uence of blood cell lysis ceeding the current analytical quality speci®cations for de- on routine coagulation testing. sirable bias were achieved for lysate concentrations of Design.ÐTwelve aliquots prepared by serial dilutions of 0.9% (prothrombin time and activated partial thrombo- homologous lysated samples collected from 10 different plastin time) and 1.8% (®brinogen), corresponding to av- subjects, and displaying a ®nal percentage of lysis ranging erage free plasma hemoglobin concentrations of 1.7 and from 0% to 9.1%, were tested for prothrombin time, ac- 3.4 g/L, respectively. tivated partial thromboplastin time, ®brinogen, and dimer- Conclusion.ÐOur results con®rm that, although slightly ized plasmin fragment D. Lysis was achieved by subjecting hemolyzed specimens might still be analyzable, a moder- citrated whole blood to a freeze-thaw cycle. ate blood cell lysis, as low as 0.9%, in¯uences the reli- Outcome Measures.ÐInterference from blood cell lysis ability of routine coagulation testing. Because the interfer- on routine coagulation testing. ence in coagulation assays has a wide interindividual bias, Results.ÐStatistically signi®cant increases in prothrom- we do not recommend lysis correction and we suggest that bin time and dimerized plasmin fragment D were observed the most appropriate corrective measure should be free in samples containing ®nal lysate concentrations of 0.5% hemoglobin quanti®cation and sample recollection. and 2.7% respectively, whereas signi®cant decreases were (Arch Pathol Lab Med. 2006;130:181±184) oagulation testing is a central aspect of the diagnostic lems arising from a cumbersome blood draw, such as un- C approach to patients with hemostasis disturbances, satisfactory attempts to draw blood, dif®culty locating and it is pivotal for monitoring antithrombotic therapies easy venous accesses, and missing the vein, are anecdot- with either heparins or oral anticoagulants.1,2 Among ma- ally known to produce major interference in routine co- jor determinants of coagulation testing, the standardiza- agulation assays, and the use of hemolyzed specimens has tion of the preanalytical phase exerts a major in¯uence on been discouraged to avoid unreliable results.4,5 However, result reliability. A standardized procedure for specimen to our knowledge, little is known about the true in¯uence collection was demonstrated to be essential to achieve ac- of unsuitable samples caused by blood cell lysis on routine curate and precise measurements, which might ®nally coagulation testing. Therefore, we evaluated the interfer- provide appropriate and suitable clinical information.3 ence of in vitro blood cell lysis on prothrombin time (PT), However, there are additional circumstances besides spec- activated partial thromboplastin time (aPTT), ®brinogen, imen collection that might in¯uence the results of coagu- and dimerized plasmin fragment D (D-dimer) testing. lation testing; these additional circumstances might also generate misleading results and induce an inappropriate MATERIALS AND METHODS diagnostic or therapeutic approach to the patients. Prob- Experiment Design and Blood Sampling On the morning of the ®rst day of the evaluation, 4.5 mL of Accepted for publication September 12, 2005. blood were separately collected in 2 siliconized vacuum tubes From the Istituto di Chimica e Microscopia Clinica, Dipartimento di containing 0.5 mL of 0.105M buffered trisodium citrate (Becton Scienze Morfologico-Biomediche, UniversitaÁ degli Studi di Verona, Ve- 3 rona, Italy. Dickinson, Oxford, United Kingdom), using a 20-gauge, 0.80 The authors have no relevant ®nancial interest in the products or 19-mm Venoject multisample straight needle (Terumo Europe companies described in this article. NV, Leuven, Belgium) from 10 healthy volunteers. Volunteers Corresponding author: Giuseppe Lippi, MD, Istituto di Chimica e gave an explicit informed consent for the investigation. Volunteers Microscopia Clinica, Dipartimento di Scienze Morfologico-Biomedi- were selected on the basis of a homogeneous range of values for che, UniversitaÁ degli Studi di Verona, Ospedale Policlinico G.B. Rossi, white blood cell counts (3.25±4.98 3 103 cells/mL), platelet count Piazzale Scuro, 10-37134ÐVerona, Italy (e-mail: [email protected]; (153±281 3 103 cells/mL), and hemoglobin concentration (137± [email protected]). 146 g/L). The ®rst specimen (sample 1) was gently mixed by Reprints not available from the authors. inverting the tube 4 to 6 times and immediately stored at 2708C, Arch Pathol Lab MedÐVol 130, February 2006 Hemolysis and Coagulation TestingÐLippi et al 181 whereas the second specimen (sample 2) was gently mixed by aPTT (Figure, B) and ®brinogen (Figure, C) values were inverting the tube 4 to 6 times and then centrifuged at 3000g for substantially decreased when compared with the baseline 8 2 8 10 minutes at 10 C. Plasma was separated and stored at 70 C. specimens (no lysate). Statistically signi®cant differences On the morning of the second day of the evaluation, blood was by the Student paired t test were observed in samples con- collected into 6 additional siliconized vacuum tubes containing 0.105M buffered trisodium citrate (Becton Dickinson) using a 20- taining a ®nal lysate concentration of 0.5% for PT, 0.9% gauge, 0.80 3 19-mm Venoject multisample straight needle (Ter- for aPTT and ®brinogen, and 2.7% for D-dimer. Percent umo) from each of the 10 volunteers. All samples were gently variations from the baseline value exceeding the current mixed by inverting the tube 4 to 6 times, pooled, and divided analytical quality speci®cations for desirable bias were into 12 aliquots of 2 mL each. Samples 1 and 2 were thawed. achieved for lysate concentrations of 0.9% (PT and aPTT) Twelve serial dilutions, obtained by mixing samples 1 and 2, and 1.8% (®brinogen), corresponding to average free plas- were prepared by adding 200 mL of each dilution to the 2-mL ma hemoglobin concentrations of 1.7 and 3.4 g/L, respec- aliquots of blood collected on the second day. Final whole blood tively. There was a roughly linear relationship between the lysate concentrations in the mixtures of samples ranged from 0% degree of variation and the percentage of lysate in the to 9.1%, roughly corresponding to average free plasma hemoglo- plasma. However, an unpredictable, sample-speci®c re- bin concentrations ranging from 0 to 17 g/L, thus, almost rep- resentative of the degree of hemolysis that we observe in speci- sponse was observed for each of the parameters assayed, mens sent to our laboratory. The blood samples were centrifuged as shown by the amplitude of the mean coef®cients of at 3000g for 10 minutes at 108C, and plasma was separated and variation, which ranged from 24% to 28% for PT, from immediately analyzed. 19% to 20% for aPTT, from 23% to 27% for ®brinogen, and from 54% to 62% for D-dimer. Because the clotting assays Laboratory Testing were performed at the alternative 570-nm wavelength, Hemolysis was assayed by measuring the concentration of free these variations were almost referable to the direct effect plasma hemoglobin by the reference cyanmethemoglobin method of hemoglobin, intracellular components, and thrombo- on a UV-1700 Spectrophotometer (Shimadzu Italia S.l.r., Milan, plastic substances released by damaged cells, rather than 6 Italy). White blood cell and platelet counts were performed on to an optical interference. an ADVIA 120 (Bayer Diagnostics, Newbury, Berkshire, United Kingdom). Routine coagulation measurements were performed COMMENT on a Behring Coagulation System (Dade-Behring, Marburg, Ger- many), using proprietary reagents: Thromborel S (lyophilized Hemolysis, causing leakage of hemoglobin and other in- human placental thromboplastin), Pathromptin SL (vegetable ternal components from the erythrocyte membrane into phospholipid with micronized silica), and Multi®bren U, for PT, the surrounding ¯uid, is usually de®ned for extracellular aPTT, and ®brinogen testing, respectively. Because hemolytic hemoglobin concentrations greater than 0.3 g/L (18.8 samples have a greater than usual inherent spectrophotometric mmol/L). Hemolysis confers a detectable pink-to-red hue absorbance in conventional clotting assays, PT, aPTT, and ®brin- to serum or plasma and becomes clearly visible in speci- ogen measurements were performed at a different wavelength mens containing as low as 0.5% hemolysate.8 Hemolyzed (570 nm), uniquely available on the BCS analyzer. Using this al- specimens are a rather frequent occurrence in laboratory ternative assay con®guration, the absorbance threshold for re- cording the coagulation time is dynamically increased by the practice, and the relative prevalence is described as being evaluation