Evaluation of the Main Coagulation Tests in the Presence of Hemolysis

International Journal of Laboratory Hematology The Ofﬁcial journal of the International Society for Laboratory Hematology

ORIGINAL ARTICLE INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY

Evaluation of the main coagulation tests in the presence of hemolysis in healthy subjects and patients on oral anticoagulant therapy G.D’ANGELO,C.VILLA,A.TAMBORINI,S.VILLA

Laboratorio di Chimica-Clinica, SUMMARY Ematologia e Microbiologia, Azienda Ospedaliera ‘S. Antonio Introduction: Our study was designed to evaluate, on healthy sub- – Abate’ Gallarate, Varese, Italy jects and patients on oral anticoagulant therapy vitamin K antago-

Correspondence: nist (OAT-vka), the possible interference caused by hemolysis on Guido D’Angelo, Ematologia/ the main coagulation tests. Coagulazione, Laboratorio di Methods: To obtain hemolyzed samples, two methods were used: Chimica-Clinica, Ematologia e heat shock and mechanical system. The coagulation tests on hemo- Microbiologia, Azienda Ospeda- – lyzed samples were performed employing optical automated analy- liera ‘S. Antonio Abate’ â Gallarate, Via Pastori 4, 21013 ser BCSxp (Siemens Healthcare ). Moreover, the prothrombin time Gallarate, Varese, Italy. (PT) and activated partial thromboplastin time (aPTT) tests were Tel.: +39 0331 751 456; also carried out manually using an electromechanical device (KC4 Fax: +39 0331 751 124; – E-mail: guido.dangelo@aogal- Amelung). larate.it Results: The PT test, on healthy subjects, in case of moderate hemolysis can be performed without significant interference on automatic instrument. On manual instrument, the PT test can be doi:10.1111/ijlh.12417 performed even in case of marked hemolysis. For patients on OAT-vka, the PT test in case of marked hemolysis can be per- Received 6 February 2015; accepted for publication 14 July formed both on automatic and manual instrument. For the aPTT 2015 test, it can be carried out manually, because also in case of marked hemolysis a statistically significant difference was not observed. Keywords For the fibrinogen test, a dramatic concentration decrease was Hemolysis, coagulation tests, already clear for weak hemolysis. A decreased function on preanalytical interference antithrombin test was statistically significant for mild-moderate hemolysis. The D-dimer test showed increased values for mild hemolysis. Conclusions: The rejection of hemolyzed sample and/or the request of a second sample are not always the proper attitudes to take for performing clotting tests. The rational management of the hemolyzed samples decreases the employment of both nursing and technical staff significantly, the turnaround time and, consequently, does not lead to additional costs for each patient involved.

Toobtainhemolyzedsamples,two methodswereused. INTRODUCTION In the first case, we used the heat shock (freezing sam- Hemolysis is characterized by the release of hemoglo- ples), in the second a mechanical system (homogenizer bin from the red blood cells and can be assessed by rotating blades). Based on the hemoglobin (Hb) amount measuring the free hemoglobin, whose limit is 0.2 in hemolyzed samples, a chromatic scale and the conse- and 0.5 g/L in plasma and serum, respectively [1]. quent ‘hemolysis index’ were defined [7]. The hemolyzed Hemolysis can occur both in vivo and in vitro; in vivo, samples were then analyzed for clotting tests using both it may be intravascular or extravascular. automatic and electromechanical manual instruments. Unsuitable procedures, due to the collection and/or management of biological samples, cause the phe- MATERIALS AND METHODS nomenon of hemolysis in vitro which affect the reliabil- ity of many tests, with the possible impact on the According to the normal reference values for white diagnosis and treatment [2]. The causes that induce blood cells count, red blood cells count, hemoglobin, pla- hemolysis in vitro may be different: the anatomical fea- telets count, C-reactive protein, and clotting tests (PT, tures of the patient, operator’s skill, the device used, aPTT, FBG, AT, and D-d), apparently healthy subjects the treatment of the sample after collection, the sample (blood donors) were recruited. The study, performed in delivery mode, the treatment of the sample before pro- accordance with the Declaration of Helsinki and under cessing, the storage of the sample. In the eventualities the terms of the local laws, did not require informed con- above listed, the haptoglobin test is not influenced by sent or Ethics Committee approval because it was carried hemolysis in vitro; therefore, to distinguish between out on samples obtained after routine analysis. hemolytic anemia in vivo and hemolysis induced On the first day of the assessment, 20 blood donors in vitro, its measurement could be used [3]. (10 males and 10 females, aged between 23 and The main mechanisms that affect the laboratory 61 years, mean age 42 years) were recruited. The blood data accuracy are three: release of elements with con- sampling was performed using 4.5-mL siliconized glass sequently false level of high intracellular concentra- tube with sodium citrate 3.2% (0.109 M) (Becton Dick- tions in serum or plasma, dilution in serum or plasma insonâ, Oxford, UK) [8]. On the first day, when the 20 of some analytes for leakage of intracellular water, samples were collected, a pool of approximately 90 mL interference in spectrophotometric determination due of anticoagulated blood was constituted. After mixing to an increase in absorbance. gently, the pool was left at room temperature for about The identification of hemolyzed samples, and con- 30 min. From the pool, three fractions of anticoagulated sequently the causes of hemolysis, is mandatory blood (aliquots A, B, and C) were constituted. The ali- because hemolysis could be a warning for significant quot A (80 mL), after being mixed gently 4–6 times, it pathologies [4]. was centrifuged for 10 min at 2700 g. The platelet-poor plasma obtained, approximately 40 mL, was the source of ‘clean plasma’ used as a control, as well as for consti- Aim and modalities of the study tuting the hemolyzed samples. It was then divided into In the context of unsuitable samples, the hemolyzed two sets of seven aliquots, each one consisting of 2 mL. samples are approximately 3% of all specimens to be The other two aliquots, B (5 mL) and C (5 mL), were tested [5], and represent 40–70% of all nonconformities used as source for preparing the hemolyzed samples. of samples [2]. In most cases, they come from the emer- The B aliquot was hemolyzed mechanically using a gency, pediatrics, and intensive care departments [6]. rotating blade homogenizer for 30 s. After centrifuga- Our study was designed to evaluate on healthy tion, the Hb concentration was measured on super- subjects (blood donors) and patients on oral anticoag- natant of aliquot B employing an automatic cell counter ulant therapy vitamin K antagonist (OAT-vka) (war- (ADVIA2120i – Siemens Healthcareâ, Siemens Health- farin), the possible interference caused by hemolysis care Diagnostics, Deerfield, IL, USA), whose hemoglobin on the main coagulation tests: prothrombin time (PT), measurement range is between 0 and 225 g/L. The ali- activated partial thromboplastin time (aPTT), fibrino- quot C, frozen at À80 °C and hemolyzed by heat shock, gen (FBG), antithrombin (AT), and D-dimer (D-d). was used for the hemolyzed samples constitution.

On the second day, after that the aliquot C was À ° Table 1. Visual evidence of hemolysis with frozen for about 24 h at 80 C, it was centrifuged corresponding hemolysis index and hemolysis for 10 min at 2700 g, and, also in this case, the Hb grading concentration on supernatant was measured. Always on the second day, the plasma set which was frozen Hemolysis Visual evidence index (g/L) Hemolysis grading at À80 °C was quickly thawed at 37 °C in a ther- mostated bath before being tested. The experimental Plasma clean Hb < 0.5 Absence of algorithm for preparing the hemolyzed samples is hemolysis – reported in Figure 1. Weakly hemolyzed Hb 0.5 1.0 Slight hemolysis sample (plasma pink) According to the ‘hemolysis index’ (Table 1, Fig- Moderately hemolyzed Hb 1.0–4.0 Moderate ure 2), the dilutions were set up taking Hb concentra- sample (plasma red) hemolysis tion of 100.0 g/L as baseline (Table 2). Markedly hemolyzed Hb > 4.0 Marked hemolysis To obtain hemolyzed samples from patients on sample (plasma from OAT-vka, the same procedure carried out for blood red to dark red) donors was used. The pool was obtained with blood samples from 20 patients, (10 males and 10 females, aged between 47 and 88 years, mean age 65 years), device (KC4 Amelung, Sigma-Amelung, GmbH- whose coagulation level was monitored periodically Lemgo, Germany). by PT–international normalized ratio (PT–INR) test. Only the PT–INR test was performed on plasma of Arbitrarily, we recruited patients whose PT–INR thera- anticoagulated patients. The analysis, also in that case, peutic range was between 2 and 3.5. was carried out on automatic and manual instruments The coagulation tests were performed on an optical simultaneously. In Table 3, the reagents and the cali- automated analyzer BCSxp (Siemens Healthcareâ, Sie- brators used, as well as the analytical methods are mens Healthcare Diagnostics Products (ex Dade- reported. Behering), Marpurg, Germany). The PT and aPTT tests were performed at two wavelengths (k), 405 and Statistical analysis 570 nm, respectively. Moreover, on hemolyzed samples from blood donors, the PT and aPTT tests were The results obtained for each test were tabulated on carried out also manually using an electromechanical Excel spreadsheet (Microsoft, Redmond, WA, USA),

Figure 1. Experimental algorithm for preparing the hemolyzed samples.

Figure 2. Chromatic variation of hemolyzed samples compared to the plasma clean (control plasma) and correlated with the ‘hemolysis index’.

Table 2. Scheme for preparing the hemolyzed RESULTS samples Prothrombin time (health subjects) Hemolyzed plasma (Hb – Plasma baseline = ‘Hemolysis index’ Mechanical hemolysis automatic system (BCSxp clean 100.0 g/L) correlated with Hb instrument) Aliquots (mL) (lL) concentration The average of hemolyzed samples compared with the 1 2 0 Plasma control (plasma average of the control plasma showed that with Hb clean) concentration up to 1.0 g/L (weak hemolysis) on auto- 2 2 10 Hemolysis weak mated instrument, k = 405 nm, there was no signifi- 3 2 20 Hemolysis weak k = 4 2 80 Moderate hemolysis cant difference (Table 4). Instead, at 570 nm, for 5 2 160 Moderate hemolysis hemolyzed samples with Hb concentration up to 4.0 g/ 6 2 320 Marked hemolysis L (moderate hemolysis), the comparison with control 7 2 640 Marked hemolysis plasma showed no statistically significant differences (Table 4). and in each table, the number of validated tests, – the mean, and the standard deviation (SD) were Mechanical hemolysis manual method reported. The results obtained for each set of hemo- [electromechanical instrument (KC4 Amelung)] lyzed samples were compared with the control Although the comparison of the mechanical system vs. plasma (clean plasma). The statistical analysis was automatic optical system showed greater imprecision performed using the variance test (ANOVA F-test); for and spread of data, the ANOVA test showed no statisti- PT and aPTT tests, only the seconds were taken into cally significant differences when we compare the account. The value of ‘critical F’ was also reported control plasma vs. hemolyzed samples with Hb values to evaluate whether between hemolyzed samples vs. up to 8.0 g/L, F = 0.935 (critical value = 4.96, control plasma there was a statistically significant P < 0.05) (Table 4). difference (P < 0.05). Moreover, arbitrarily, we con- sidered the threshold of 3% below which interfer- Prothrombin time (health subjects) ence with the hemolysis was not significant. We believe that the 3% threshold value is a good com- Heat shock hemolysis – automatic system (BCSxp promise between the intra- and interindividual bio- instrument) logical variability. Moreover, it does not significantly affect the clinical end-point for diagnostic and thera- The average of hemolyzed samples compared with the peutic purposes [9–11]. average of the control plasma showed that already

Table 3. Instruments, reagents, calibrators and method used

Tests Instrument Kit reagent Calibrator Analytical method

PT BCSxp Innovin PT multicalibrator Coagulative (Siemens Healthcareâ) (Siemens Healthcareâ) (Siemens Healthcareâ) PT KC4 Innovin Not used* Coagulative (Amelung) (Siemens Healthcareâ) aPTT BCSxp Actin FS Not used* Coagulative (Siemens Healthcareâ) (Siemens Healthcareâ) aPTT KC4 Actin FS Not used* Coagulative (Amelung) (Siemens Healthcareâ) Fibrinogen BCSxp Multiﬁbren U Fibrinogen calibrator kit Coagulative (Siemens Healthcareâ) (Siemens Healthcareâ) (Siemens Healthcareâ) Antithrombin BCSxp AT III Standard human plasma Colorimetric (Siemens Healthcareâ) antithrombin (Siemens Healthcareâ) (Siemens Healthcareâ) D-dimer BCSxp Innovance Innovance D-dimer calibrator Turbidimetric (Siemens Healthcareâ) D-dimer (Siemens Healthcareâ) (Siemens Healthcareâ)

PT, prothrombin time; aPTT, activated partial thromboplastin time. *Pool plasma in house as reference value. considering a weak hemolysis (Hb = 1.0 g/L) on auto- k = 570 nm, the difference vs. control plasma becomes mated instrument, k = 405 nm, the ANOVA test showed significant for marked hemolysis with Hb concentra- a significant difference, F = 6.79 (critical value = 4.96, tion more than 8.0 g/L (Table 5). P < 0.05) (Table 4). Even with k = 570 nm, the ANOVA test showed a statistically significant difference with Mechanical hemolysis – manual method Hb = 1.0 g/L (weak hemolysis), F = 9.918 (critical [electromechanical instrument (KC4 Amelung)] value = 4.96, P < 0.05) (Table 4). In this case, the ANOVA test showed statistically significant difference with moderate hemolysis (Hb = 4.0 g/ Heat shock hemolysis – manual method L), F = 8.045 (critical value = 4.96, P < 0.05) (Table 5). [electromechanical instrument (KC4 Amelung)]

In this case, comparing the hemolyzed plasma vs. con- Heat shock hemolysis – automatic system (BCSxp trol plasma, the difference became statistically signifi- instrument) cant with samples markedly hemolyzed (Hb = 8.0 g/L), F = 10.29 (critical value = 4.96, P < 0.05) (Table 4). On automated instrument, k = 405 nm, comparing the data with the control plasma, the difference became statistically significant with weak hemolysis Prothrombin time –patients on oral anticoagulant (Hb = 0.5 g/L), F = 12.25 (critical value = 4.96, therapy vitamin K antagonist P < 0.05) (Table 5). Again with weak hemolysis (Hb = 0.5 g/L), at k = 570 nm, the difference became Mechanical hemolysis – automatic system (BCSxp statistically significant (Table 5). instrument)

On automatic system, k = 405 nm, the ANOVA test Heat shock hemolysis – manual method showed that the difference vs. control plasma became [electromechanical instrument (KC4 Amelung)] statistically signiﬁcant with weak hemolysis (Hb = 1.0 g/L), F = 18.223 (critical value = 4.96, Comparing the data with the control plasma, the P < 0.05) (Table 5). Always on automatic system, ANOVA test showed that with Hb concentration up to

Table 4. Prothrombin time test analysis on normal subjects. Heat shock and mechanical hemolysis. Mean, standard deviation, F critic, and variation (D) report. Automatic system (BCSxp instrument) and manual method [Electromechanical instrument (KC4 Amelung)] TA.|HMLSSITREEC NCAUAINTESTING COAGULATION ON INTERFERENCE HEMOLYSIS | AL. ET BCSxp k = 405 nm BCSxp k = 570 nm Electromechanical device (KC4 Amelung)

Validated Mean Validated Mean Validated Mean tests (s) SD F D (%) tests (s) SD F D (%) tests (s) SD F D (%)

Heat shock hemolysis Pool 6 8.917 0.075 6 9.033 0.052 6 9.783 0.331 Hb 6 9.000 0.126 1.923 0.9 6 9.150 0.084 8.448 1.3 6 9.967 0.197 1.360 1.9 0.5 g/L Hb 6 9.117 0.172 6.792 2.2 6 9.217 0.133 9.918 2.0 6 10.067 0.151 3.640 2.9 1.0 g/L Hb 6 33.017 17.006 12.050 270.3 6 9.367 0.163 22.727 3.7 6 10.050 0.122 3.422 2.7 4.0 g/L Hb 6 42.217 23.559 11.987 373.4 6 9.583 0.214 37.552 6.1 6 10.400 0.335 10.293 6.3 8.0 g/L Hb 5 62.133 34.107 18.237 597.9 6 10.500 0.341 108.764 16.2 6 10.183 0.496 2.702 4.1 16.0 g/L Hb 4 44.683 17.041 40.854 401.0 5 32.720 28.956 5.038 262.2 5 10.350 0.259 10.906 5.8 © 32.0 g/L 05Jh ie osLtd, Sons & Wiley John 2015 Mechanical hemolysis Pool 6 9.067 0.234 6 9.333 0.266 6 10.183 0.360 Hb 6 9.167 0.175 0.703 1.1 6 9.233 0.103 0.738 À1.1 6 9.900 0.341 1.961 À2.8 0.5 g/L Hb 6 9.200 0.179 1.231 1.5 6 9.283 0.117 0.178 À0.5 6 9.978 0.461 0.737 À2.0 1.0 g/L Hb 6 33.550 33.208 3.261 270.0 6 9.467 0.137 1.194 1.4 6 10.050 0.259 0.542 À1.3 4.0 g/L n.Jl a.Hem. Lab. Jnl. Int. Hb 6 47.167 32.120 8.442 420.2 6 9.800 0.190 12.250 5.0 6 10.333 0.121 0.935 1.5 8.0 g/L Hb 5 62.000 38.046 11.877 583.8 6 10.683 0.313 64.960 14.5 6 10.583 0.172 6.025 3.9 16.0 g/L Hb 6 40.867 34.416 5.122 350.7 3 13.567 4.446 n.c. 45.4 6 10.583 0.349 3.820 3.9

2015, 32.0 g/L 37 819–833 , © 05Jh ie osLtd, Sons & Wiley John 2015

Table 5. Prothrombin time test analysis on OAT-(vka) patients. Heat shock and mechanical hemolysis. Mean, standard deviation, F critic, and variation (D) report. Automatic system (BCSxp instrument) and manual method [Electromechanical instrument (KC4 Amelung)]

BCSxp k = 405 nm BCSxp k = 570 nm Electromechanical device (KC4 Amelung)

n.Jl a.Hem. Lab. Jnl. Int. Validated Mean Validated Mean Validated Mean tests (s) SD F D (%) tests (s) SD F D (%) tests (s) SD F D (%)

Heat shock hemolysis Pool 6 15.217 0.075 6 15.333 0.137 6 16.583 0.331 Hb 6 14.967 0.121 12.250 À1.6 6 15.150 0.105 6.798 À1.2 6 16.883 0.354 2.295 1.8 2015, 0.5 g/L Hb 6 14.883 0.271 8.403 À2.2 6 15.233 0.175 1.216 À0.7 6 17.583 0.436 20.045 6.0 37

819–833 , 1.0 g/L .DANGELO G. Hb 6 57.583 41.765 6.174 278.4 6 15.317 0.133 0.046 À0.1 6 17.617 0.306 31.508 6.2 4.0 g/L Hb 4 47.075 34.096 5.587 209.4 4 15.767 0.163 24.853 2.8 6 17.550 0.339 24.955 5.8 8.0 g/L

Hb 3 56.367 36.153 9.069 270.4 6 16.767 0.367 80.391 9.4 6 17.233 0.383 9.889 3.9 TESTING COAGULATION ON INTERFERENCE HEMOLYSIS | AL. ET 16.0 g/L Hb 3 62.100 32.419 14.640 308.1 5 17.580 2.980 2.817 14.7 6 17.817 0.483 26.583 7.4 32.0 g/L Mechanical hemolysis Pool 6 15.267 0.103 6 15.400 0.297 6 15.883 0.256 Hb 6 15.133 0.163 2.857 À0.9 6 15.267 0.273 0.656 À0.9 6 15.683 0.214 2.156 À1.3 0.5 g/L Hb 6 14.917 0.172 18.223 À2.3 6 15.200 0.200 1.875 À1.3 6 16.133 0.163 4.061 1.6 1.0 g/L Hb 6 13.850 3.658 0.899 À9.3 6 15.283 0.075 0.872 À0.8 6 16.283 0.232 8.045 2.5 4.0 g/L Hb 4 62.775 14.946 40.484 311.2 4 15.633 0.151 2.952 1.5 6 16.517 0.436 9.426 4.0 8.0 g/L Hb 6 51.883 33.739 7.067 239.8 6 16.233 0.175 35.112 5.4 6 16.617 0.417 13.482 4.6 16.0 g/L Hb 5 54.660 27.764 10.069 258.0 5 17.233 0.151 182.229 11.9 6 16.900 0.335 34.906 6.4 32.0 g/L

OAT-(vka), oral anticoagulant therapy vitamin K antagonist. 825 826 G. DANGELO ET AL. | HEMOLYSIS INTERFERENCE ON COAGULATION TESTING

0.5 g/L (weak hemolysis), there was no significant dif- Heat shock hemolysis ference, F = 2.295 (critic value = 4.96, P < 0.05) (Table 5). As statistically shown by the test ‘F’, the analysis of hemolyzed samples with heat shock showed high spreading of the data with Hb concentration up to Activated partial thromboplastin time (healthy subjects) 4.0 g/L (moderate hemolysis) (Table 7). Mechanical hemolysis – automatic system (BCSxp instrument) Antithrombin The analysis of the data for both wavelengths, Mechanical hemolysis k = 405 nm and k = 570 nm, respectively, showed that there was no statistically significant difference for The analysis of variance between hemolyzed samples Hb up to 16.0 g/L (Table 6). vs. control plasma showed a statistically significant difference with Hb = 1.0 g/L (weak hemolysis) (Table 8). Mechanical hemolysis – manual method [electromechanical instrument (KC4 Amelung)] Heat shock hemolysis

The data obtained on our manual instrument in this The ANOVA showed that with moderate hemolysis case showed that there was no statistically signiﬁcant (Hb = 4.0 g/L), statistically, there was no signiﬁcant difference for Hb up to 32.0 g/L (Table 6). difference between hemolyzed samples with heat shock vs. control plasma (Table 8). Heat shock hemolysis – automatic system (BCSxp instrument) D-dimer

On automatic instrument, k = 405 nm, comparing the Comparing both sets of hemolyzed samples, mechani- data with the control plasma, the difference became cal and heat shock, respectively, with control plasma, statistically significant already considering a weak the ANOVA showed a significant difference already for hemolysis (Hb = 0.5 g/L), while the analysis of data at mild hemolysis (Hb = 0.5 g/L) (Table 9). wavelength k = 570 nm showed that the difference vs. control plasma was not statistically significant for DISCUSSION Hb values up to 16.0 g/L (Table 6). The clotting tests are the ones most affected by the different pre-analytical variables. In case of hemolysis, Heat shock hemolysis – manual method the main constituents able to interfere with the clot- [electromechanical instrument (KC4 Amelung)] ting process are not yet known. The red blood cell The data obtained employing our manual instrument damage leads not only to the leakage of hemoglobin showed that, statistically, there was no significant dif- from the cytoplasm, but generates also circulating ference for Hb values up to 16.0 g/L (Table 6). cell-derived microparticles that, activating the coagulation factors, speed up the coagulation pathway [12]. In addition to hemoglobin, other substances that Fibrinogen interfere with the clotting process can be released from platelets and/or leukocytes. Mechanical hemolysis The relationship between the level of hemoglobin Comparing the data with the control plasma, as statis- present in the sample hemolyzed and the degree of tically demonstrated by ANOVA, significant differences interference on the coagulation testing, if any, is com- were already observed for hemolyzed samples with plex. Hemoglobin present on supernatant does not Hb = 0.5 g/L (Table 7). seem to affect the clotting test because it is

Table 6. Activated partial thromboplastin time test analysis on normal subjects. Heat shock and mechanical hemolysis. Mean, standard deviation, F critic, and variation (D) report. Automatic system (BCSxp instrument) and manual method [Electromechanical instrument (KC4 Amelung)]

BCSxp k = 405 nm BCSxp k = 570 nm Electromechanical device (KC4 Amelung) n.Jl a.Hem. Lab. Jnl. Int. Validated Mean Validated Mean Validated Mean tests (s) SD F D (%) tests (s) SD F D (%) tests (s) SD F D (%)

Heat shock hemolysis Pool 6 29.167 0.472 6.360 6 29.917 0.496 6 27.967 0.484 À

2015, Hb 6 30.233 0.922 8.013 3.7 6 31.000 0.732 9.009 3.6 6 27.883 0.556 0.077 0.3 0.5 g/L

37 Hb 6 30.433 0.989 11.212 4.3 6 30.167 0.615 1.187 0.8 6 28.133 0.501 0.343 0.6 819–833 ,

1.0 g/L DANGELO G. Hb 6 40.633 8.375 3.573 39.3 6 29.833 0.819 0.036 À0.3 6 27.833 0.615 0.174 À0.5 4.0 g/L Hb 6 42.950 13.351 6.387 47.3 6 29.483 0.924 0.315 À1.4 6 27.533 0.408 2.807 À1.6 8.0 g/L Hb 6 47.700 27.448 2.735 63.5 6 32.167 2.538 5.048 7.5 6 28.367 0.599 1.618 1.4 TESTING COAGULATION ON INTERFERENCE HEMOLYSIS | AL. ET 16.0 g/L Hb 6 55.600 34.799 3.461 90.6 6 43.233 8.284 15.779 44.5 6 28.733 0.680 5.057 2.7 32.0 g/L Mechanical hemolysis Pool 6 29.800 0.932 6 29.800 0.696 6 27.617 0.585 Hb 6 29.883 0.519 0.037 0.3 6 30.233 0.393 1.764 1.5 6 27.617 0.426 0.100 0.0 0.5 g/L Hb 6 30.117 0.736 0.427 1.1 6 30.433 0.747 2.308 2.1 6 27.517 0.214 0.155 À0.4 1.0 g/L Hb 6 32.300 9.799 0.387 8.4 6 30.167 0.568 1.000 1.2 6 27.167 0.520 1.984 À1.6 4.0 g/L Hb 6 34.617 10.316 1.298 16.2 6 29.733 1.041 0.017 À0.2 6 27.000 0.537 3.624 À2.2 8.0 g/L Hb 5 35.017 30.232 0.178 17.5 5 28.717 1.550 2.440 À3.6 6 26.967 0.873 2.296 À2.4 16.0 g/L Hb 6 32.917 8.171 0.862 10.5 6 34.233 3.779 7.986 14.9 6 27.317 0.471 0.959 À1.1 32.0 g/L 827 828 G. DANGELO ET AL. | HEMOLYSIS INTERFERENCE ON COAGULATION TESTING

Table 7. Fibrinogen test analysis on normal subjects. Heat shock and mechanical hemolysis. Mean, standard deviation, F critic, and variation (D) report. Automatic system (BCSxp instrument)

Heat shock hemolysis Mechanical hemolysis

Validated Mean Validated Mean tests (mg/dL) SD F D (%) tests (mg/dL) SD F D (%)

Pool 6 301.500 4.764 6 256.833 12.336 Hb 6 292.500 8.118 5.485 À3.0 6 290.833 9.475 28.669 13.2 0.5 g/L Hb 6 289.833 16.510 2.766 À3.9 6 297.500 8.044 45.755 15.8 1.0 g/L Hb 5 209.400 135.075 2.336 À30.5 5 195.800 110.312 1.496 À23.8 4.0 g/L Hb 3 99.000 23.065 234.236 À67.2 4 196.500 115.049 1.016 À23.5 8.0 g/L Hb 6 218.167 75.798 7.224 À27.6 6 230.167 214.063 0.093 À10.4 16.0 g/L Hb 4 146.750 125.651 6.263 À51.3 4 223.250 224.366 0.080 À13.1 32.0 g/L

Table 8. Antithrombin test analysis on normal subjects. Heat shock and mechanical hemolysis. Mean, standard deviation, F critic, and variation (D) report. Automatic system (BCSxp instrument)

Heat shock hemolysis Mechanical hemolysis

Validated Mean Validated Mean tests (%) SD F D (%) tests (%) SD F D (%)

Pool 6 100.000 1.095 6 98.167 4.622 Hb 6 99.833 4.070 0.507 À0.2 6 94.833 1.472 2.833 À3.4 0.5 g/L Hb 6 101.000 4.561 0.273 1.0 6 93.167 2.927 5.011 À5.1 1.0 g/L Hb 6 100.833 1.722 1.000 0.8 6 92.500 2.881 6.494 À5.8 4.0 g/L Hb 6 95.000 2.000 28.846 À5.0 6 90.667 2.251 12.768 À7.6 8.0 g/L Hb 6 90.333 2.251 89.468 À9.7 6 84.167 3.189 37.294 À14.3 16.0 g/L Hb 6 81.333 4.761 87.598 À18.7 6 76.333 2.066 111.580 À22.2 32.0 g/L

stroma-free, unlike the whole hemolysate that, as part be extended because the phospholipidic membranes of the stroma, contains microvesicles. The anionic can compete with the thromboplastin for the avail- phospholipidic membranes exposed during the hemo- ability of FVII activated and thus lead to the lengthen- lytic process can increase the intake of phospholipids, ing of the clotting time [13]. For coagulation testing, providing a thrombogenic surface to the activation it is not possible to predict unambiguously how the and propagation of the coagulation cascade. This analytical data will be affected. would explain the possible shortening of clotting time. Reiterating that the difference (D) below 3% However, it is possible also that the clotting time can (D < 3%) between hemolyzed plasma vs. control

Table 9. D-dimer test analysis on normal subjects. Heat shock and mechanical hemolysis. Mean, standard deviation, F critic, and variation (D) report. Automatic system (BCSxp instrument)

Heat shock hemolysis Mechanical hemolysis

Validated Mean Validated Mean tests (ng/mL FEU) SD F D (%) tests (ng/mL FEU) SD F D (%)

Pool 6 254.833 9.988 6 280.000 8.173 Hb 6 270.667 7.312 9.816 6.2 6 321.667 12.738 49.63 14.9 0.5 g/L Hb 6 274.333 11.039 10.294 7.7 6 324.000 13.266 45.474 15.7 1.0 g/L Hb 6 304.333 11.237 65.041 19.4 6 329.167 11.089 47.842 17.6 4.0 g/L Hb 6 377.667 14.801 283.936 48.2 6 354.167 21.894 76.432 26.5 8.0 g/L Hb 6 509.667 15.616 1133.883 100.0 6 417.500 27.588 60.429 49.1 16.0 g/L Hb 6 686.333 67.808 237.812 169.3 6 473.833 43.282 137.018 69.2 32.0 g/L

plasma does not interfere on the diagnostic and instrument (Amelung – KC4), even in case of therapeutic end-point, the study, based on data marked hemolysis (Hb up to 16.0 g/L), the PT test analysis, gave us the following operative indications. can be performed on samples hemolyzed with both For the PT test, on healthy subjects, in case of mod- methods (Figure 3). For patients on OAT-vka, even erate hemolysis (both mechanical and heat shock in case of marked hemolysis (Hb up to 8.0 g/L), on and Hb up to 4.0 g/L), the test can be performed both hemolyzed samples (mechanical and heat on automatic instrument at k = 570 nm without sig- shock), the PT test, run on automatic instrument, niﬁcant interference (Figure 3). On our manual k = 570 nm, as well as on our electromechanical

Figure 3. Prothrombin time test trend on normal subjects considering D Æ 3%. (1) Heat shock hemolysis – BCSxp k = 405 nm ( ); (2) heat shock hemolysis – BSCxp k = 570 nm ( ); (3) heat shock hemolysis – KC4 Amelung ( ); (4) mechanical hemolysis – BCSxp k = 405 nm ( ); (5) mechanical hemolysis – BCSxp k = 570 nm ( ); (6) mechanical hemolysis – KC4 Amelung ( ).

manual instrument (Amelung – KC4), showed a dif- data were reliable with k = 405 nm and k = 570 nm, ference D ≤ 3% when compared to control plasma respectively. (Figure 4). For the FBG test, the analysis compared with the For the aPTT test (Figure 5), the data showed that control plasma statistically showed a significant it could be appropriate to carry out the test manually difference with weak hemolysis; a dramatic concentra- because also in case of marked hemolysis (Hb up to tion decrease was clear for Hb = 0.5 g/L (weak hemol- 32.0 g/L) with both methods, a statistically significant ysis) (Figure 6). difference was not observed. With automatic instru- For the AT test, a decreased function of the glyco- ment, only in case of mild hemolysis (Hb up to 1.0 g/ protein was statistically significant for mild–moderate L) and moderate hemolysis (Hb up to 8.0 g/L), the hemolysis (Figure 7).

Figure 4. Prothrombin time test trend on OAT patients considering D Æ 3%. (1) Heat shock hemolysis – BCSxp k = 405 nm ( ); (2) heat shock hemolysis – BSCxp k = 570 nm ( ); (3) heat shock hemolysis – KC4 Amelung ( ); (4) mechanical hemolysis – BCSxp k = 405 nm ( ); (5) mechanical hemolysis – BCSxp k = 570 nm ( ); (6) mechanical hemolysis – KC4 Amelung ( ).

Figure 5. Activated partial thromboplastin time test trend on normal subjects considering D Æ 3%. (1) Heat shock hemolysis – BCSxp k = 405 nm ( ); (2) heat shock hemolysis – BSCxp k = 570 nm ( ); (3) heat shock hemolysis – KC4 Amelung ( ); (4) mechanical hemolysis – BCSxp k = 405 nm ( ); (5) mechanical hemolysis – BCSxp k = 570 nm ( ); (6) mechanical hemolysis – KC4 Amelung ( ).

For the D-dimer test, the data, when compared hemolyzed samples with visible hemolysis and Hb up with the control plasma, showed a statistically signiﬁ- to 4.0 g/L can be processed for PT and aPTT tests on cant increase for mild hemolysis (Figure 8). automatic instrument at k = 570 nm. However, each Although the analyzed data of our study are laboratory should identify the most reliable threshold related to a surrogate hemolysis, we showed that the of interference by hemolysis according to local rejection of hemolyzed sample, and/or the request of reagents and instruments used [15]. a second sample, are not always the proper attitudes The rational management of the hemolyzed sam- to take for performing clotting tests. In agreement ples decreases the employment of both nursing and with Arora et al. [14], by which samples with visible technical staff signiﬁcantly, as well as the ‘inconve- hemolysis can be processed for the most common nience’ on the patient, the turnaround time and, con- coagulation tests (PT and aPTT), also in our case, sequently, reduces the additional costs for each

Figure 6. Fibrinogen test trend on normal subjects considering D Æ 3%. BCSxp instrument. , mechanical hemolysis; , heat shock hemolysis.

Figure 7. Antithrombin test trend on normal subjects considering D Æ 3%. BCSxp instrument. , mechanical hemolysis; , heat shock hemolysis.

Figure 8. D-dimer test trend on normal subjects considering D Æ 3%. BCSxp instrument. , heat shock hemolysis; , mechanical hemolysis.

patient involved [16]. Moreover, in case of hemolysis, cine, Academic Hospital of Parma, Parma, Italy) for his variations of a few seconds, which are statistically sig- valuable suggestions on important aspects of this study. nificant on PT test, often do not have a clinical impact We thank the hematological technical staff (Mrs Carla in patients on OAT-vka [13]. Banfi, Mr Giuseppe Carrozzo, Mrs Maria Rosa Further, larger sample size studies must be carried Colombo, Mrs Maria Carla Guatta Cescone, Mrs Ivana out to confirm the data of pilot studies, especially for Lonido, Mrs Ornella Varallo) for having contributed to patients on heparin and oral anticoagulant therapy, in the implementation of the study. particular for oral direct inhibitors (dabigatran, rivaroxaban, apixaban, or edoxaban). AUTHORSHIP

GD, CV, AT, SV performed, designed the research ACKNOWLEDGEMENTS study, analyzed the data, revising the manuscript and We would like to thank Prof. Giuseppe Lippi (Director approval for the ﬁnal version. GD wrote the manu- and Associate Professor Laboratory of Clinical-Chem- script. istry and Hematology, Department of Laboratory Medi-

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