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The Effect of 3.2% and 3.8% Sodium Citrate on Specialized Coagulation Tests.

Despite a high degree of standardization, coagulation tests largely depend on preanalytic factors. Apart from artifacts acquired during blood drawing and transportation, the in vitro anticoagulant used has a potential impact on the coagulation test result. Usually, a 9:1 blood to sodium citrate ratio is used with a buffer range between 3.2% and 3.8%. The 3.2% buffered sodium citrate binds less assay-added calcium than 3.8% buffered sodium citrate, therefore clotting times tend to be shorter in 3.2% than 3.8% buffered sodium citrate. (1) Although there are no exact data available, today 3.2% buffered citrate is rather widely used. A survey conducted in 2001 revealed that 156 of 593 US hospitals (26%) used the 3.8% buffered citrate for prothrombin time (PT) analysis. (2) The 3.8% sodium citrated plasma appears to alter the International Sensitivity Index (ISI) of some thromboplastin times (3-5) but is still the preferred material for special platelet function tests. For practical reasons, each institution usually uses 1 citrate concentration level for both coagulation testing and platelet function testing. Most recommendations are based on studies evaluating various PT and activated partial thromboplastin time (aPTT) reagents. (6,7) The buffer concentration might amplify the effect of preanalytic factors, including the hematocrit or underfilling or overfilling of collecting tubes. Here, 3.2% buffered citrate tubes might be less influenced by underfilling than 3.8% buffered tubes during assessment of the global assays aPTT or PT. (3,8-10)

However, precise knowledge about their impact on specialized coagulation tests is lacking. Overwhelming amounts of studies were conducted with these 2 buffer concentration ranges. To provide comparative data, we conducted this observational study for estimating the effects on a broad panel of coagulation tests as well as on their reference value ranges.

MATERIALS AND METHODS

Subjects

This prospective observational study was conducted at the Medical University of Vienna (Vienna, Austria) between September 2015 and July 2016. After we obtained written informed consent, 76 apparently healthy adult volunteers were included in the study. Volunteers undergoing therapy with anticoagulants (within the last 30 days) or with known coagulation disorders were excluded from participation. The study was approved by the local ethics committee (No. 404/2011) and was conducted in accordance with the 1964 Declaration of Helsinki (including current revisions). Blood samples were taken with 21-gauge needles in siliconized tubes (Vacuette, Greiner Bio-One, Kremsmiinster, Austria) prefilled with trisodium citrate (3.2% or 3.8%). After gentle tilting, the samples were centrifuged at 2500g for 15 minutes at 15[degrees]C (Hettich Mikro 220R, Tuttlingen, Germany). For assays of the lupus anticoagulant panel the samples were centrifuged a second time under the same conditions to obtain platelet-free citrate plasma. Coagulation analyses were performed either by using freshly drawn specimens or once-thawed aliquots stored below -70[degrees]C.

Methods

All analyses were executed under standardized conditions at the Department of Laboratory Medicine, which maintains a certified (according to ISO [International Organization for Standardization] 9001:2008) and accredited (according to ISO 15189:2008) quality management system. Using the same calibration settings, 3.2% and 3.8% buffered citrate samples were consecutively analyzed. Table 1 represents an overview of the analyzer platforms, reagents, calibrators, and controls used. The reagent-analyzer combinations were chosen with respect to measurement accuracy and validity, assays' robustness and practicability during the parameters' introduction phase at our laboratory. All assay procedures were performed according to the manufacturer's recommendations. Automatic multidilution procedures were applied for factor testing. The aPTT-LA (lupus-sensitive reagent) for lupus anticoagulant (LAC) confirmation testing was analyzed by using plasma with and without a reagent containing phosphatidylethanolamine. Lupus anticoagulant confirmation testing was considered positive when the difference between both aPTT-LA clotting times was greater than 8 seconds ([aPTT-LA.sub.Difference], Staclot LA [Diagnostica Stago S.A.S, Asnieres sur Seine, France]) or the ratio between the neat diluted Russell viper venom time (dRVVT) and the [dRVVT.sub.Confirm] ([dRVVT.sub.Ratio], Staclot DRVV Screen/Staclot DRVV Confirm) was greater than 1.25.

Statistical Analysis

Numeric values are given as median with the interquartile range and are analyzed with the Wilcoxon rank sum test and the Spearman rank correlation test ([r.sub.[rho]]). The Passing-Bablok regression analysis was applied with bootstrapped confidence intervals. (11,12) To ensure linearity between 2 tested parameters, a CuSum test was applied. Further, Bland-Altman plots are used to assess agreement between the 2 methods (3.2% and 3.8% citrate) and scatterplots are applied to assess the linearity between the 2 methods. (13) To establish the reference interval, occurrence of Gaussian distribution (Shapiro-Wilk test) and the symmetry of skewness were assessed. Outliers were detected with the Tukey test. (14) If necessary, a logarithm transformation or a Box-Cox transformation was performed. (15) Reference intervals were calculated by using the robust method in accordance with the Clinical and Laboratory Standards Institute guideline C28-A3, (16) which uses a bootstrapping approach. Statistical significance was defined as P values less than .05 (2-tailed). Where appropriate, the Bonferroni-Holm method was applied to adjust for an error related to multiple testing. Data were statistically analyzed by using MedCalc Version 16.8.4 (Ostend, Belgium).

RESULTS

Among the 21 coagulation assays evaluated, the following 6 tests presented a significant prolongation or activity reduction when using 3.8% buffered citrate compared to 3.2% buffered citrate: aPTT-FS (factor-sensitive reagent containing ellagic acid); [dRVVT.sub.Confirm]; [dRVVT.sub.Ratio]; and activity levels of factor V, factor VIII, and protein C (Table 2). These parameters are graphically presented by using Bland-Altman plots (Figure 1, a through f) and their respective indicators of comparability are shown in Table 3. In Passing-Bablok regression analysis, a systemic and proportional difference was found in the aPTT-FS (Figure 2) with 3.80 intercept (0.24-7.49) and 0.86 slope (0.76-0.96). All other parameters with a significant difference did not show a systemic or proportional difference.

Lupus anticoagulant confirmation testing is based on calculated parameters and therefore potential measurement errors might be boosted. The [aPTT-LA.sub.Difference] indicated no significant difference between both citrate concentrations, using the Mann-Whitney U test. Passing-Bablok regression analysis was not applicable, since a significant deviation of linearity was found (Figure 3, a and b). However, the rank correlation coefficient was in a moderate range ([r.sub.[rho]] = 0.511), displaying a proportional difference between both citrate concentrations. In a next step, we sought to establish whether the use of different citrate concentrations led to any alterations of reference intervals. Most coagulation assays presented only minor differences in reference intervals between both citrate buffer concentrations (Table 4). Nonoverlapping 90% confidence intervals between 3.2% and 3.8% buffered citrate samples were found in the aPTT-FS. Lupus anticoagulant confirmation tests showed comparable reference range limits.

DISCUSSION

Despite the practical importance, limited comparative data are available for specialized coagulation test results measured with 3.2% and 3.8% buffered citrate samples. Most coagulation parameters tested showed good comparability between both citrate buffer concentrations. The aPTT-FS presented with a systematic and proportional deviation and therefore the citrate buffer concentration had a significant impact on this measurement. Further, the citrate buffer concentration significantly affected the upper reference range limit of the aPTT-FS. In comparison to the aPTT-A (activated partial thromboplastin time), a pronounced effect of the buffer concentration on the aPTT-FS was also found by Adcock et al. (6)

One might speculate that calculated coagulation parameters are more sensitive to alterations of the citrate buffer concentration, since 2 or more factors are used, each having a potential error itself. The [aPTT-LA.sub.Difference] presented only a moderate correlation, with a systemic difference between both citrate concentrations. Therefore, we advise special caution when comparing data of aPTT-LA confirmation testing. Interestingly, this effect was not seen in dRVVT confirmation testing. However, the [dRVVT.sub.Confirm] clotting time was considerably shortened in the higher citrate buffer concentration, contrary to expectations. In none of the cases tested was a false-positive result detected in either [dRVVT.sub.Ratio] or [aPTT-LA.sub.Difference] confirmatory testing. Furthermore, a statistically significant difference was found in [dRVVT.sub.Confirm], [dRVVT.sub.Ratio], factor V, factor VIII, and protein C activity, but their absolute difference was in a minor range and no deviation could be detected in regression analysis.

In the literature, there are several publications reporting a significant influence of the citrate concentration on the aPTT or PT, (6,7,17) but also publications that do not find any alteration in regard to the citrate concentration used. Apart from the parameter of inherent susceptibility to alterations of the citrate concentration, this might indicate that different reagent-analyzer combinations have different sensibilities in regard to the citrate concentrations used. (3,17,18) For 60 patients receiving oral anticoagulants, the relative difference of the PT between both buffer concentrations ranged from 3.7% to 20% when measured under similar conditions using different reagent-analyzer combinations. (17) Aside from the sodium concentration, the amount of magnesium or other electrolytes within the buffer might also have a significant influence on the comparability of parameters. (19,20) Along these lines, the tube brand has a statistically significant influence on the results of the PT or aPTT, as shown for 5 different tube brands containing 3.2% buffered citrate. (20,21)

In this evaluation, we assessed a broad spectrum of coagulation parameters by using a single specific reagent-analyzer combination for each parameter (Table 1). The reagent-analyzer combination itself might have an impact on the measurement, and therefore our results are not generalizable for other settings. (3,17,18) Since we evaluated healthy volunteers without any coagulation disorders or anticoagulant therapy, we are not able to estimate the potential difference in pathologic samples. The data regarding the influence of the citrate buffer concentration on monitoring anticoagulation are nebulous. Adcock et al (6) found in 5% of examined samples a significant deviation in INR (international normalized ratio) determination, due to different citrate buffer concentrations. Further, Payne et al (22) did not observe any statistically significant influence of the citrate concentration on low-molecular-weight heparin monitoring in patients with pulmonary embolism or deep vein thrombosis.

In conclusion, the alteration of 3.2% and 3.8% buffered sodium citrate on specialized coagulation tests in our setting was of limited relevance. As an exception, the aPTT-FS and [aPTT-LA.sub.Difference] were significantly affected by the citrate concentration used. Our findings demonstrate the comparability of data assessed with these citrate concentration ranges.

We extend our thanks to John Heath for his attentive proofreading of our manuscript.

References

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(6.) Adcock DM, Kressin DC, Marlar RA. Effect of 3.2% vs 3.8% sodium citrate concentration on routine coagulation testing. Am J Clin Pathol. 1997;107(1):105- 110.

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(8.) Koepke JA, Rodgers JL, Ollivier MJ. Pre-instrumental variables in coagulation testing. Am J Clin Pathol. 1975;64(5):591-596.

(9.) Reneke J, Etzell J, Leslie S, Ng VL, Gottfried EL. Prolonged prothrombin time and activated partial thromboplastin time due to underfilled specimen tubes with 109 mmol/L (3.2%) citrate anticoagulant. Am J Clin Pathol. 1998;109(6): 754-757.

(10.) Adcock DM, Kressin DC, Marlar RA. Minimum specimen volume requirements for routine coagulation testing: dependence on citrate concentration. Am J Clin Pathol. 1998;109(5):595-599.

(11.) Bilic-Zulle L. Comparison of methods: passing and Bablok regression. Biochem Med (Zagreb). 2011;21(1):49-52.

(12.) Passing H, Bablok. A new biometrical procedure for testing the equality of measurements from two different analytical methods: application of linear regression procedures for method comparison studies in clinical chemistry, part I. J Clin Chem Clin Biochem. 1983;21(11):709-720.

(13.) Giavarina D. Understanding Bland Altman analysis. Biochem Med (Zagreb). 2015;25(2):141-151.

(14.) Tukey JW. Exploratory Data Analysis. 1st ed. Boston: Addison-Wesley Publishing Company; 1977.

(15.) Sakia RM. The Box-Cox transformation technique: a review. Statistician. 1992;41(2):169-178.

(16.) Ozarda Y. Reference intervals: current status, recent developments and future considerations. Biochem Med (Zagreb). 2016;26(1):5-11.

(17.) Chantarangkul V, Tripodi A, Clerici M, Negri B, Mannucci PM. Assessment of the influence of citrate concentration on the International Normalized Ratio (INR) determined with twelve reagent-instrument combinations. Thromb Haemost. 1998;80(2):258-262.

(18.) Lottin L, Woodhams BJ, Saureau M, et al. The clinical relevance of the citrate effect on International Normalized Ratio determinations depends on the reagent and instrument combination used. Blood Coagul Fibrinolysis. 2001;12(5): 399-404.

(19.) van den Besselaar AMHP, van Vlodrop IJH, Berendes PB, Cobbaert CM. A comparative study of conventional versus new, magnesium-poor Vacutainer[R] Sodium Citrate blood collection tubes for determination of prothrombin time and INR. Thromb Res. 2014;134(1):187-191.

(20.) van den Besselaar A, Hoekstra M, Witteveen E, Didden JH, van der Meer FJM. Influence of blood collection systems on the prothrombin time and international sensitivity index determined with human and rabbit thromboplastin reagents. Am J Clin Pathol. 2007;127(5):724-729.

(21.) Lima-Oliveira G, Lippi G, Salvagno GL, Montagnana M, Picheth G, Guidi GC. Sodium citrate vacuum tubes validation: preventing preanalytical variability in routine coagulation testing. Blood Coagul Fibrinolysis. 2013;24(3):252-255.

(22.) Payne S, MacKinnon K, Keeney M, Morrow B, Kovacs MJ. Effect of 3.2 vs. 3.8% sodium citrate concentration on anti-Xa levels for patients on therapeutic low molecular weight heparin. Clin Lab Haematol. 2003;25(5):317-319.

Franz Ratzinger, MD; Mona Lang; Sabine Belik; Klaus G. Schmetterer, MD, PhD; Helmuth Haslacher, MD; Thomas Perkmann, MD; Peter Quehenberger, MD

Accepted for publication October 23, 2017.

Published as an Early Online Release April 6, 2018.

From the Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria.

The authors have no relevant financial interest in the products or companies described in this article.

Corresponding author: Peter Quehenberger, MD, Department of Laboratory Medicine, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria (email: peter.quehenberger@ meduniwien.ac.at).

Caption: Figure 1. Bland-Altman plots of selected parameters using 3.2% and 3.8% citrate. a, aPTT-FS (ellagic acid containing activated partial thromboplastin time reagent). b, [dRVVT.sub.Confirm] (diluted Russell viper venom time confirmation testing). c, [dRVVT.sub.Ratio] (ratio between the neat dRVVT and the [dRVVT.sub.Confirm]). d, Factor V. e, Factor VIII. f, Protein C activity.

Caption: Figure 2. Scatterplot of the aPTT-FS using 3.2% and 3.8% citrate. Abbreviation: aPTT-FS, ellagic acid containing activated partial thromboplastin time reagent.

Caption: Figure 3. Graphical assessment of the [aPTT-LA.sub.Difference] using 3.2% and 3.8% citrate. a, Bland-Altman plot. b, Scatterplot. Abbreviations: aPTT-LA, activated partial thromboplastin time lupus-sensitive reagent; [aPTT-LA.sub.Difference], difference between clotting times of the aPTT-LA with and without a phosphatidylethanolamine-containing reagent.
Table 1. Overview of Methods Used for Assessment

Method                     Unit           Analyzer

Prothrombin time             s       STA-R Evolution (a)
Fibrinogen               mg/dL (c)   STA-R Evolution (a)
aPTT-A                       s       STA-R Evolution (a)
aPTT-LA                      s       STA-R Evolution (a)
aPTT-FS                      s       STA-R Evolution (a)
dRVVT                        s       STA-R Evolution (a)
[dRVVT.sub.Confirm]          s       STA-R Evolution (a)
[aPTT-LA.sub.Confirm]        s       MC10 PLUS (d)
Antithrombin III             %       STA-R Evolutiona
vWF-antigen                  %       STA-R Evolutiona
Factor II, factor V          %       Sysmex CA-7000 (b)
Factor VII                   %       Sysmex CA-7000 (b)
Factor X                     %       Sysmex CA-7000 (b)
Factor Vlll/factor IX        %       Sysmex CA-7000 (b)
Factor XI                    %       Sysmex CA-7000 (b)
Factor XII                   %       Sysmex CA-7000 (b)
Protein C activity           %       ACL-TOP (e)
Protein S activity           %       STA-R Evolution (a)
Protein S antigen            %       ACL-TOP (e)

Method                                     Reagents

Prothrombin time         Thromborel S (b)
Fibrinogen               STA-Liquid Fib (a)
aPTT-A                   STA-aPTT (a)
aPTT-LA                  PTT-LA (a)
aPTT-FS                  Dade Actin FS aPTT (b)
dRVVT                    Staclot DRVV Screen (a)
[dRVVT.sub.Confirm]      Staclot DRVV Confirm (a)
[aPTT-LA.sub.Confirm]    PTT-LA (with phospholipids) (a) Staclot LA
Antithrombin III         Stachrom ATIII (a)
vWF-antigen              Sta Liatest VWF Ag (a)
Factor II, factor V      Thromborel S (b), deficient plasma (b)
Factor VII               Thromborel S (b), deficient plasma (b)
Factor X                 Thromborel S (b), deficient plasma (e)
Factor Vlll/factor IX    Dade Actin FS aPTT (b), deficient plasma (f)
Factor XI                Dade Actin FS aPTT (b), deficient plasma (b)
Factor XII               Dade Actin FS aPTT (b), deficient plasma (b)
Protein C activity       Biophen Protein C5 (g)
Protein S activity       Staclot Protein S (a)
Protein S antigen        Free Protein S (e)

Method                             Calibrators

Prothrombin time         Standard Human Plasma (b)
Fibrinogen                             NA
aPTT-A                                 NA
aPTT-LA                                NA
aPTT-FS                                NA
dRVVT                                  NA
[dRVVT.sub.Confirm]                    NA
[aPTT-LA.sub.Confirm]                  NA
Antithrombin III         STA Unicalibrator (a)
vWF-antigen              VWF-Ag Calibrator (a)
Factor II, factor V      STA Unicalibrator (a)
Factor VII               Standard Human Plasma (b)
Factor X                 STA Unicalibrator (a)
Factor Vlll/factor IX    STA Unicalibrator (a)
Factor XI                STA Unicalibrator (a)
Factor XII               Standard Human Plasma (b)
Protein C activity       STA Unicalibrator (a)
Protein S activity       STA Unicalibrator (a)
Protein S antigen        HemosIL Calibration Plasma (e)

Abbreviations: aPTT-A, activated partial thromboplastin time; aPTT-FS,
ellagic acid containing activated partial thromboplastin time reagent;
aPTT-LA, lupus-sensitive activated partial thromboplastin time;
[aPTT-LA.sub.Confirm], lupus-sensitive activated partial thromboplastin
time confirmation testing; dRVVT, diluted Russell viper venom time;
[dRVVT.sub.Confirm], diluted Russell viper venom time confirmation
testing; NA, not applicable; vWF, von Willebrand factor antigen.

(a) Diagnostica Stago S.A.S, Asnietres sur Seine, France.

(b) Siemens Healthcare GmbH, Erlangen, Germany.

(c) Divide by 100 for g/L.

(d) ABW Medizin und Technik GmbH, Lemgo, Germany.

(e) Werfen, Barcelona, Spain.

(f) Technolclone GmbH, Vienna, Austria.

(g) Hyphen BioMed, Neuville-sur-Oise, France.

Table 2. Synopsis of the Alteration Caused by the Citrate Buffer
Concentration

                                3.2% Citrate   3.8% Citrate
Parameter                  N    Median (IQR)   Median (IQR)

PT                         76   13.1 (0.7)      12.9 (0.8)
Fibrinogen                 78    266 (82)      260.5 (77)
Antithrombin III           77    104 (9.3)       104 (9.5)
vWF-antigen                73     95 (38)         93 (35.5)
aPTT-A                     78   34.5 (3.4)      34.6 (3.6)
aPTT-FS                    77     35 (2.7)      36.5 (3.6)
aPTT-LA                    79   38.2 (4)        37.9 (4.7)
[aPTT-LA.sub.Confirm]      63   40.2 (4.3)      40.9 (4.4)
[aPTT-LA.sub.Difference]   63    2.4 (2.7)       1.5 (2.7)
dRVVT                      70   42.6 (4.4)      43.3 (5.3)
[dRVVT.sub.Confirm]        63   41.5 (3.5)      40.3 (4.4)
[dRVVT.sub.Ratio]          62   1.04 (0.09)     1.06 (0.1)
Factor II                  76    110 (23)        110 (24)
Factor V                   76    115 (24)        112 (27)
Factor VII                 76     96 (29)         96 (27)
Factor X                   76    110 (26)        109 (27)
Factor VIII                76    128 (64)        128 (61)
Factor IX                  76    113 (33)        112 (31)
Factor XI                  76     95 (20)         96 (18)
Factor XII                 76    112 (35)        111 (34)
Protein C activity         75    101 (23)        100 (22)
Protein S activity         73    108 (26)        110 (28)
Protein S antigen          76    104 (29)        103 (27)

                            Wilcoxon      Correlation
Parameter                  P Value (a)   [r.sub.[rho]]

PT                             .06           0.897
Fibrinogen                     .10           0.943
Antithrombin III               .56           0.894
vWF-antigen                    .10           0.933
aPTT-A                         .16           0.921
aPTT-FS                     <.001 (b)        0.801
aPTT-LA                        .08           0.920
[aPTT-LA.sub.Confirm]          .01           0.769
[aPTT-LA.sub.Difference]       .09           0.515
dRVVT                          .02           0.864
[dRVVT.sub.Confirm]         <.001 (b)        0.927
[dRVVT.sub.Ratio]           <.001 (b)        0.714
Factor II                      .89           0.982
Factor V                    <.001 (b)        0.969
Factor VII                     .63           0.983
Factor X                       .82           0.917
Factor VIII                 <.001 (b)        0.973
Factor IX                      .02           0.975
Factor XI                      .40           0.933
Factor XII                     .12           0.984
Protein C activity          <.001 (b)        0.978
Protein S activity             .62           0.940
Protein S antigen              .74           0.974

                               Passing-Bablok Regression

Parameter                   Intercept                 Slope

PT                         -1.00 (-16.00 to 8.40)   1.00 (0.90-1.17)
Fibrinogen                 -2.44 (-14.96 to 9.89)   1.01 (0.97-1.07)
Antithrombin III           0.00 (-10.20 to 9.45)    1.00 (0.90-1.10)
vWF-antigen                 2.03 (-1.69 to 6.09)    0.98 (0.94-1.03)
aPTT-A                      0.20 (-2.47 to 1.96)    1.00 (0.95-1.08)
aPTT-FS                     3.80 (0.24 to 7.49)     0.86 (0.76-0.96)
aPTT-LA                     1.71 (-1.50 to 4.69)    0.96 (0.88-1.04)
[aPTT-LA.sub.Confirm]       2.26 (-5.11 to 8.60)    0.96 (0.80-1.14)
[aPTT-LA.sub.Difference]      Significant deviation from linearity
dRVVT                       3.76 (-1.37 to 8.29)    0.90 (0.79-1.03)
[dRVVT.sub.Confirm]         1.99 (-1.83 to 5.61)    0.97 (0.88-1.07)
[dRVVT.sub.Ratio]           0.02 (-0.19 to 0.21)    0.96 (0.77-1.11)
Factor II                   1.53 (0.00 to 7.07)     0.98 (0.93-1.00)
Factor V                    -0.382 (-5.7 to 3.7)    0.97 (0.93-1.00)
Factor VII                  0.00 (-1.82 to 4.07)    1.00 (0.96-1.02)
Factor X                    0.00 (0.00 to 4.64)     1.00 (0.96-1.00)
Factor VIII                -0.95 (-6.37 to 4.02)    1.02 (0.99-1.07)
Factor IX                  -2.41 (-10.44 to 3.53)   1.04 (0.97-1.11)
Factor XI                   0.00 (-4.99 to 5.29)    1.00 (0.95-1.05)
Factor XII                 -0.77 (-4.87 to 0.00)    1.01 (1.00-1.05)
Protein C activity          1.00 (-2.08 to 6.50)    1.00 (0.95-1.04)
Protein S activity         1.60 (-6.94 to 10.30)    0.98 (0.90-1.06)
Protein S antigen           0.80 (-3.21 to 3.71)    1.00 (0.97-1.04)

Abbreviations: aPTT-A, activated partial thromboplastin time; aPTT-FS,
ellagic acid containing activated partial thromboplastin time reagent;
aPTT-LA, lupus sensitive activated partial thromboplastin time;
[aPTT-LA.sub.Confirm], lupus sensitive activated partial thromboplastin
time confirmation testing; [aPTT-LA.sub.Diference], lupus sensitive
activated partial thromboplastin time confirmation testing difference;
dRVVT, diluted Russell viper venom time; [dRVVT.sub.Confirm], diluted
Russell viper venom time confirmation testing; [dRVVT.sub.Ratio],
diluted Russell viper venom time confirmation testing ratio; IQR,
interquartile range;PT, prothrombin time;vWF, von Willebrand factor
antigen.

(a) All P values are given 2-sided.

(b) Statistical siginficance after Bonferroni-Holm correction.

Table 3. Synopsis of Bland-Altman Plot Indices

                              Mean ([+ or -] SD)
Method                Unit       3.2% Citrate

aPTT-FS               s       34.8 ([+ or -] 2.2)
[dRVVT.sub.Confirm]   s       41.2 ([+ or -] 2.7)
[dRVVT.sub.Ratio]     ...     1.04 ([+ or -] 0.08)
Factor V              %      113.4 ([+ or -] 19.5)
Factor VIII           %      142.0 ([+ or -] 45.8)
Protein C activity    %      103.1 ([+ or -] 16.4)

                       Mean ([+ or -] SD)
Method                    3.8% Citrate            Bias (95% CI)

aPTT-FS                36.3 ([+ or -] 2.5)      -1.5 (-1.8 to -1.2)
[dRVVT.sub.Confirm]    40.4 ([+ or -] 2.8)       0.9 (0.6 to 1.1)
[dRVVT.sub.Ratio]      1.07 ([+ or -] 0.08)    -0.03 (-0.05 to -0.02)
Factor V              110.9 ([+ or -] 18.3)      2.4 (1.2 to 3.7)
Factor VIII           138.6 ([+ or -] 42.8)      3.4 (1.4 to 5.4)
Protein C activity    101.6 ([+ or -] 16.6)      1.4 (0.7 to 2.2)

                         Lower Limit of          Upper Limit of
Method                 Agreement (95% CI)      Agreement (95% CI)

aPTT-FS                -4.0 (-4.6 to -3.5)        1.1 (0.6-1.6)
[dRVVT.sub.Confirm]    -1.1 (-1.6 to -0.7)        2.9 (2.4-3.3)
[dRVVT.sub.Ratio]     -0.14 (-0.17 to -0.12)     0.08 (0.06-0.10)
Factor V               -8.2 (-10.3 to -6.1)      13.1 (10.9-15.2)
Factor VIII           -13.6 (-17.1 to -10.2)     20.4 (17.0-23.8)
Protein C activity     -4.8 (-6.1 to -3.6)        7.7 (6.4-9.0)

Abbreviations: aPTT-FS, ellagic acid containing activated partial
thromboplastin time reagent; [dRVVT.sub.Confirm], diluted Russell
viper venom time confirmation testing; [dRVVT.sub.Ratio], diluted
Russell viper venom time confirmation testing ratio.

Table 4. Reference Range (Lower and Upper Limit)
of Assessed Parameters (a,b)

Parameter                  N      3.2% Citrate       3.8% Citrate

Prothrombin time           76   12.1 (12.0-12.3)   12.1 (11.9-12.2)
                                14.2 (13.9-14.3)   14.1 (13.8-14.2)
Fibrinogen                 78    162 (146-178)      164 (149-179)
                                 375 (357-391)      368 (349-384)
Antithrombin III           77      89 (87-92)         89 (87-91)
                                 118 (116-121)      119 (116-121)
vWF-antigen                73      57 (54-62)         56 (52-60)
                                 176 (157-196)      178 (158-201)
aPTT-A                     78   29.5 (28.8-30.3)   29.5 (28.8-30.2)
                                39.4 (38.5-40.3)   39.4 (38.5-40.2)
aPTT-FS                    77   30.7 (29.9-31.5)   31.3 (30.5-32.1)
                                39.1 (38.5-39.8)   41.3 (40.5-42.1)
aPTT-LA                    79   32.8 (32.1-33.7)   32.1 (31.2-33.0)
                                44.0 (43.0-44.9)   44.3 (43.2-45.3)
[aPTT-LA.sub.Confirm]      63   34.2 (33.1-35.5)   33.8 (32.7-35.0)
                                47.1 (45.9-48.4)   46.1 (45.0-47.1)
[aPTT-LA.sub.Difference]   63    Not performed      Not performed
                                 6.6 (5.8-7.4)      6.2 (5.3-7.0)
dRVVT                      70   35.4 (34.3-36.6)   35.6 (34.4-36.8)
                                49.5 (48.2-50.8)   51.1 (49.7-52.3)
[dRVVT.sub.Confirm]        63   35.9 (34.9-36.9)   34.9 (33.9-36.0)
                                46.9 (46.0-47.7)   46.2 (45.3-47.1)
[dRVVT.sub.Ratio]          62    Not performed      Not performed
                                1.18 (1.14-1.20)   1.22 (1.19-1.25)
Factor II                  76      80 (75-85)         79 (74-84)
                                 143 (139-148)      142 (136-148)
Factor V                   76      76 (69-83)         75 (69-81)
                                 151 (145-157)      148 (143-153)
Factor VII                 76      58 (51-64)         57 (50-63)
                                 135 (128-142)      135 (128-142)
Factor X                   76      73 (66-79)         72 (65-79)
                                 149 (142-155)      149 (142-155)
Factor VIII                76      71 (65-79)         72 (65-79)
                                 242 (214-269)      241 (216-265)
Factor IX                  76      75 (70-80)         75 (70-80)
                                 169 (158-181)      163 (152-173)
Factor XI                  76      65 (60-70)         68 (64-73)
                                 123 (118-129)      122 (118-127)
Factor XII                 76      63 (55-73)         66 (59-75)
                                 157 (150-166)      154 (148-162)
Protein C activity         75      75 (71-78)         72 (69-76)
                                 136 (129-143)      137 (129-145)
Protein S activity         73      66 (59-73)         65 (59-73)
                                 153 (145-160)      153 (145-160)
Protein S antigen          76      65 (59-71)         64 (59-71)
                                 140 (135-145)      140 (135-146)

Abbreviations: aPTT-A, activated partial thromboplastin time; aPTT-FS,
ellagic acid containing activated partial thromboplastin time reagent;
aPTT-LA, lupus-sensitive activated partial thromboplastin time;
[aPTT-LA.sub.Confirm], lupus-sensitive activated partial thromboplastin
time confirmation testing; [aPTT-LA.sub.Difference], lupus-sensitive
activated partial thromboplastin time confirmation testing difference;
dRVVT, diluted Russell viper venom time; [dRVVT.sub.Confirm], diluted
Russell viper venom time confirmation testing; [dRVVT.sub.Ratio],
diluted Russell viper venom time confirmation testing ratio; vWF, von
Willebrand factor antigen.

(a) Lower limit/upper limit.

(b) Outliers were detected with the Tukey test.
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Author:Ratzinger, Franz; Lang, Mona; Belik, Sabine; Schmetterer, Klaus G.; Haslacher, Helmuth; Perkmann, Th
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Report
Date:Aug 1, 2018
Words:4571
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