Clinica Chimica Acta 442 (2015) 49–51

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Letter to the Editor Matrix and bilirubin interference for high-sensitivity cardiac troponin I

To the Editor: Pre-analytical factors may adversely affect high-sensitivity cardiac troponin results. In particular, we and others have demonstrated the need for appropriate centrifugation to mitigate falsely elevated highsensitivity cardiac troponin I (hs-cTnI) results on stored samples [1,2]. The package insert for the Abbott Architect hs-cTnI (REF 3P25, Revised September 2013) assay does include a thorough section on appropriate centrifugation protocols; however, it is unclear how these protocols were derived. Moreover, a standard centrifugation protocol to be assessed by all manufacturers for cTn would be extremely useful for laboratories when comparing and contemplating offering hs-cTnI testing. This aspect has been recently opined by Apple et al. with respect to key elements that should be addressed and standardized in study protocols for manufacturers of cardiac troponin assays [3]. One element that was not addressed in this excellent opinion piece, and typically has minimal information provided in the package inserts for cTnI assays, is on endogenous interferences. For cardiac troponin, haemolysis has been shown to adversely affect both cTnT and cTnI measurements, with the extent and magnitude of this interference being different between the cTnI manufacturers [4]. With the increased analytical sensitivity and precision that are characteristic of highsensitivity cardiac troponin assays [2,3], the need to evaluate interferences in the matrix which is used for clinical purposes is of upmost importance if the goal is to prevent misinterpretation of a change in cardiac troponin concentration due to the presence of an interference. For the Abbott Architect hs-cTnI assay, the initial Australian validation publication on the assay assessed various amounts of haemoglobin across different hs-cTnI concentrations, and identified that haemoglobin N5 g/l resulted in an underestimation of hs-cTnI concentration; however this study did not assess bilirubin [5]. As our laboratory was evaluating the Architect hs-cTnI assay, we assessed two common plasma sample types for hs-cTn testing (EDTA plasma and lithium heparin plasma) for potential bilirubin interferences at different cardiac troponin I concentrations with both the Architect hs-cTnI assay and Architect contemporary cTnI assay. Briefly, bilirubin (Sigma, 25 mmol/l of stock solution frozen and protected from light) was spiked into either EDTA patient-pooled plasma or lithium heparin patient-pooled plasma (2 different concentrations) to achieve the different bilirubin concentrations. A concentration of bilirubin was characterized as an interference if it surpassed the manufacturer's allowable difference as stated in the package inserts. The allowable limits for deviations acceptable for bilirubin interference was different between the two assays, with the contemporary assay allowing up to 15% difference, and the hs-cTnI assay permitting 10% difference. This was followed by another experiment by spiking the concentration of bilirubin yielding the interference across several

http://dx.doi.org/10.1016/j.cca.2015.01.002 0009-8981/© 2015 Elsevier B.V. All rights reserved.

different individual patient samples (n = 20 paired EDTA and lithium heparin plasma) to identify the hs-cTnI concentration that was affected with the bilirubin interference. In order to perform this experiment, 31 paired EDTA and lithium heparin plasma patient samples were measured to identify 20 suitable pairs for the subsequent spiking analyses. Passing and Bablok, ordinary linear regressions, and difference plots were performed via Analyse-it (ver 2.30) software, with 95% CI calculated with p b 0.05 considered significant. With the contemporary cTnI assay, based on an imprecision (coefficient of variation) of 20% at concentrations close to the 99th percentile, there was no noticeable interference with bilirubin in either EDTA plasma or lithium heparin plasma (see Table 1, N2.7 SD used to set upper and lower limits of acceptance per reference [5]). At higher cTnI concentrations (Fig. 1), bilirubin appeared to negatively affect the cTnI concentrations in both EDTA plasma and lithium heparin plasma, but were within the allowable 15% difference as per the contemporary assay's specifications. Assessing the hs-cTnI assay, only high bilirubin concentrations (N 342 μmol/l) appeared to negatively affect the hs-cTnI concentrations in the EDTA pooled samples whereas bilirubin concentrations N200 μmol/l appeared to decrease hs-cTnI concentrations in the lithium heparin plasma pools (i.e., ≥ 10% difference; see Fig. 1). Thirty-one paired patient EDTA and lithium heparin plasma samples were measured for hs-cTnI; with lithium heparin plasma samples yielding significantly higher results (lithium heparin plasma [hs-cTnI] = 1.08 (95% CI: 1.04 to 1.11) EDTA plasma[hs-cTnI] + 0.18 ng/l (95% CI; −0.32 to 0.86)) with the average percent positive bias being 7.1% across the full range (Fig. 2 a). Focusing on the concentrations b150 ng/l (n = 28), where absolute differences (i.e., change in concentrations or the delta) may be a more important variable, there is a small absolute positive bias between lithium heparin plasma and EDTA plasma (3.3 ng/l (95% CI: 0.9 to 5.8); p = 0.009), with lithium heparin hs-cTnI concentrations N10 ng/l higher than EDTA plasma hs-cTnI concentrations mostly at concentrations N 60 ng/l (Fig. 2 b).

Table 1 The effect of bilirubin on cardiac troponin I results close to the 99th percentile with the contemporary assay in EDTA plasma and lithium heparin plasma. Matrix

cTnI (μg/l)

Low EDTA pool neat Low EDTA pool spike 1 Low EDTA pool spike 2 Low EDTA pool spike 3 Low EDTA pool spike 4 Low lithium heparin pool neat Low lithium heparin pool spike 1 Low lithium heparin pool spike 2 Low lithium heparin pool spike 3 Low lithium heparin pool spike 4

0.034 0.037 0.037 0.049 0.042 0.051 0.042 0.046 0.047 0.051

Acceptable range (μg/l) Low

Bilirubin (μmol/l)

I-Index

13 384 211 113 64 14 399 211 114 65

14 484 245 121 65 17 519 264 135 74

High

0.016 0.052 Within Within Within Within 0.024 0.079 Within Within Within Within

50

Letter to the Editor

Fig. 1. The effect of bilirubin on cardiac troponin I recovery in different matrices (lithium heparin plasma pooled samples and EDTA plasma pooled samples) measured by a high-sensitivity assay and contemporary assay.

Of the 31 samples, 20 samples were selected to be spiked with bilirubin to yield N342 μmol/l for EDTA plasma (average (range) bilirubin concentration = 354 μmol/l (348–364)) with the matching lithium heparin samples spiked with bilirubin to yield N200 μmol/l (average (range) bilirubin concentration = 213 μmol/l (206–225)). There were 2 samples that had hs-cTnI concentrations N1000 ng/l which yielded an average decrease of 15% when spiked with the specific bilirubin interference (in agreement with the high pool results in Fig. 1). Assessing the absolute decrease in hs-cTnI concentrations b 150 ng/l (n = 18), there appears to be a linear trend for both EDTA and lithium heparin plasma (Fig. 3). Graphically, hs-cTnI concentrations N 60 ng/l were most likely to yield decreases of more than 10 ng/l when the bilirubin interference was present (Fig. 3). These data indicate that the extent of the bilirubin interference for hs-cTnI measurements is dependent on the concentration of bilirubin, the concentration of hs-cTnI, and the sample type. As myocardial injury is clinically evaluated in various hospitalized populations outside of the emergency department (ED), it may be prudent for laboratories to consider appending comments for haemolysed

a

and icteric samples. In our particular hospital, approximately half of the cardiac troponin orders are from the ED. Interestingly, within the first week of offering hs-cTnI testing, the prevalence of icteric EDTA plasma samples (I-index N342) with hs-cTnI measurements, which were generally from patients in the oncology and intensive care units, was 1.3%. This was higher than the prevalence of haemolysed samples (H-index N5) at 0.6%, which were mainly from ED patients. The increased rate of haemolysed samples arriving from the ED (i.e., via collections through IV catheters) has been well documented and the potential effect that haemolysis has on cardiac troponin interpretation [6,7]. There are mechanisms that can be employed to lessen the amount of haemolysed samples [7]; however, this cannot be done for high bilirubin concentrations/icteric samples. Our study is noteworthy, in that it confirms other reports suggesting that there is significant difference in hs-cTnI concentrations when measured in lithium heparin versus EDTA plasma [8,9]. Specifically, hs-cTnI concentrations are higher in lithium heparin plasma and typically are ≥10 ng/l in lithium heparin versus EDTA plasma at hs-cTnI concentrations N60 ng/l. This difference exceeds the 10 ng/l limit as suggested

b Percent Difference Plot

Absolute Difference Plot 25

20% 20 15%

Difference (Lithium Heparin - EDTA)

Difference (Lithium Heparin - EDTA) / Mean of hs-cTnI (ng/L)

25%

10% 5% 0% -5% -10%

15

10

5

0

-5 -15% -10

-20% 0

500

1000

1500

2000

2500

Identity

Allowable bias (10%)

Bias (7.1%)

0

20

40

60

80

100

120

140

Mean of hs-cTnI (ng/L)

Mean of hs-cTnI (ng/L) 95% CI

Identity

Bias (3.3)

95% CI

Fig. 2. Difference plot between hs-cTnI measured in lithium heparin plasma vs hs-cTnI measured in EDTA plasma: a) percent difference across range of samples; b) absolute difference for hs-cTnI concentrations b150 ng/l.

Letter to the Editor

Absolute Difference Plot EDTA with Bilirubin Interference (>342 umol/L) 5

0

-5

-10 y = -0.17x + 0.6 R2 = 0.80

-15

-20

b Difference (Li Heparin with Bilirubin (213 umol/L) - Li Heparin)

Difference (EDTA with Bilirubin (354 umol/L) - EDTA)

a

-25

51

Absolute Difference Plot lithium heparin with Bilirubin Interference (>200 umol/L) 5

0

-5

-10 y = -0.13x + 1.5 R2 = 0.7424

-15

-20

-25 0

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

0

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

Mean of hs-cTnI (ng/L) Identity

Bias (-7.2 ng/L)

95% CI

Mean of hs-cTnI (ng/L) Linear

Identity

Bias (-5.0 ng/L)

95% CI

Linear

Fig. 3. Absolute difference plot for hs-cTnI concentrations with bilirubin N342 μmol/l (average = 354 μmol/l; range: 348–364 μmol/l) in EDTA plasma (a) and hs-cTnI concentrations with bilirubin N200 μmol/l (average = 213 μmol/l; range: 206–225 μmol/l) in lithium heparin plasma (b).

by Simpson et al. [10], and re-enforces the importance of using only one type of matrix for hs-cTnI measuring and reporting. Moreover, it would appear that high bilirubin concentrations also cause a significant absolute decrease in hs-cTnI concentrations also N 60 ng/l; however the amount of bilirubin needed to cause this significant decrease in hscTnI concentrations is different between lithium heparin (bilirubin N200 μmol/l) and EDTA (bilirubin N 342 μmol/l) plasma. Common criteria used by all manufacturers assessing endogenous interferences would be useful when assessing and interpreting high-sensitivity cardiac troponin assays/results. Moreover, interference testing should be performed on all matrices supported by the manufacturer so that laboratories can decide on the best sample type to mitigate inaccurate cardiac troponin results due to endogenous interferences.

References [1] Kavsak PA, Caruso N, Beattie J, Clark L. Centrifugation—an important pre-analytical factor for the Abbott Architect high-sensitivity cardiac troponin I assay. Clin Chim Acta 2014;436:273–5. [2] Ryan JB, Southby SJ, Stuart LA, Mackay R, Florkowski CM, George PM. Comparison of cardiac TnI outliers using a contemporary and high-sensitivity assay on the Abbott Architect platform. Ann Clin Biochem 2014;51:507–11. [3] Apple FS, Hollander J, Wu AH, Jaffe AS. Improving the 510(k) FDA process for cardiac troponin assays: in search of common ground. Clin Chem 2014;60:1273–5. [4] Florkowski C, Wallace J, Walmsley T, George P. The effect of hemolysis on current troponin assays—a confounding preanalytical variable? Clin Chem 2010;56:1195–7. [5] Koerbin G, Tate J, Potter JM, Cavanaugh J, Glasgow N, Hickman PE. Characterisation of a highly sensitive troponin I assay and its application to a cardio-healthy population. Clin Chem Lab Med 2012;50:871–8. [6] Kavsak PA, Worster A. Dichotomizing high-sensitivity cardiac troponin T results and important analytical considerations. J Am Coll Cardiol 2012;59:1570. [7] Kavsak PA. A step closer in reducing hemolysis in blood samples collected in the emergency department. Clin Biochem 2013;46:565.

[8] Kavsak PA, MacRae AR, Yerna MJ, Jaffe AS. Analytic and clinical utility of a nextgeneration, highly sensitive cardiac troponin I assay for early detection of myocardial injury. Clin Chem 2009;55:573–7. [9] Krintus M, Kozinski M, Boudry P, Capell NE, Köller U, Lackner K, et al. European multicenter analytical evaluation of the Abbott ARCHITECT STAT high sensitive troponin I immunoassay. Clin Chem Lab Med 2014;52:1657–65. [10] Simpson AJ, Potter JM, Koerbin G, Oakman C, Cullen L, Wilkes GJ, et al. Use of observed within-person variation of cardiac troponin in emergency department patients for determination of biological variation and percentage and absolute reference change values. Clin Chem 2014;60:848–54.

Janet Simons Lori Beach Department of Pathology and Molecular Medicine McMaster University, Hamilton, ON, Canada Lorna Clark Juravinski Hospital and Cancer Centre, Hamilton Health Sciences, Hamilton, ON, Canada Peter A. Kavsak Department of Pathology and Molecular Medicine McMaster University, Hamilton, ON, Canada Juravinski Hospital and Cancer Centre, Hamilton Health Sciences, Hamilton, ON, Canada Corresponding author at: Juravinski Hospital and Cancer Centre, 711 Concession Street Hamilton, ON L8V 1C3, Canada. Tel.: +1 905 521 2100. E-mail address: [email protected]. 10 December 2014

Matrix and bilirubin interference for high-sensitivity cardiac troponin I.

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