Artificially depleted plasmas are not necessarily commutable with native patient plasmas for International Sensitivity Index calibration and International Normalized Ratio derivation: a reply to a rebuttal


Anton M. H. P. van den Besselaar, Department of Thrombosis and Hemostasis, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, the Netherlands.
Tel.: +31 71 526 1894; fax: +31 71 526 6755.


See also Poller L, Jespersen J, Ibrahim S. Artificially depleted plasmas are not necessarily commutable with native patient plasmas for International Sensitivity Index calibration and International Normalized Ratio derivation: a rebuttal. This issue, p 1197–8; van den Besselaar AMHP. Artificially depleted plasmas are not necessarily commutable with native patient plasmas for International Sensitivity Index calibration and International Normalized Ratio derivation. J Thromb Haemost 2012; 10: 303–5.

With interest, I read the letter by Poller et al. With respect to this letter, I wish to raise several points.

In 2004, ‘Guidelines on preparation, certification, and use of certified plasmas for ISI calibration and INR determination’ were published on behalf of the Subcommittee on Control of Anticoagulation of the Scientific and Standardization Committee of the ISTH [1]. According to these guidelines, each set or batch of certified plasmas intended for either local test system International Sensitivity Index (ISI) calibration or direct International Normalized Ratio (INR) determination must be validated before release. The validation should be the responsibility of the manufacturer or supplier. The validation should go through the following process: (i) 10 or more fresh plasmas from patients treated with vitamin K antagonists (VKAs) are selected to represent the full therapeutic range of anticoagulation; (ii) the INR of these fresh plasmas shall be determined with an appropriate international standard for thromboplastin, and the mean value (INRR) shall be calculated; (iii) the INR of the same fresh plasmas shall also be determined with a variety of commercial reagent/instrument combinations, following the certified plasma procedure, and the mean value (INRC) shall be calculated; and (iv) paired INR values obtained with the international standard and with the commercial system are compared to assess their agreement, by the use of Bland and Altman’s procedure. If the relative difference between the INRR and INRC is 10% or less, the set of certified plasmas are considered to be acceptable, and may be released for local ISI calibration or direct INR determination.

The European Concerted Action on Anticoagulation (ECAA) has prepared plasmas by artificial depletion of normal human plasma by selective adsorption of vitamin K-dependent clotting factors with barium sulfate. In one study, calibration with artificially depleted plasmas was compared to calibration with fresh VKA plasmas, using only one particular combination of thromboplastins, i.e. a human brain thromboplastin and human recombinant thromboplastin [2]. In this study, differences were observed between the calibration lines obtained with the two types of plasma, but were regarded as acceptable in clinical terms. The results of a related study suggested that certified prothrombin time values for freeze-dried plasmas cannot be used for ISI calibration of dissimilar thromboplastins [3]. Subsequently, many ECAA studies have been published on laboratory system calibrations with lyophilized artificially depleted plasmas, using widely different thromboplastins (e.g. prepared from rabbit brain, human placenta, or bovine brain), but in none of these studies were comparisons made with fresh VKA plasmas [4]. Even in the most recent ECAA publications on the PT/INR line, validation of the artificially depleted plasmas with fresh VKA plasmas was not performed [5]. In the latter ECAA studies, INR values of lyophilized ‘validation’ plasmas were compared before and after correction with the PT/INR line. The INR deviations of ‘validation’ plasmas from certified values were reduced with sets of human, bovine and rabbit reagents. This is not surprising, because the artificially depleted ‘validation’ plasmas had been prepared in the same way as the artificially depleted ‘calibrant’ plasmas.

Why are artificially depleted plasmas not necessarily commutable with fresh plasmas of patients treated with VKA? [6]. The answer to this question may be found in an analysis of the clotting factor composition of these plasmas. Table 1 shows the mean activities of factor II, FVII and FX in plasmas derived from patients treated with VKA, in comparison with the mean values measured in plasmas artificially depleted of clotting factors by variable chemical adsorption. In plasmas from VKA-treated patients, FVII activity is significantly higher than FX activity [7–10]. In contrast, in artificially depleted plasmas, FVII activity is significantly lower than FX activity [11]. Thromboplastin reagents can vary widely in their sensitivity to reductions in the levels of vitamin K-dependent clotting factors. It is now apparent that dissimilar thromboplastin compositions are associated with dissimilar relative sensitivities to deficiencies in FII, FV, FVII, and FX, particularly as clotting factor deficiencies become more severe [12]. It seems likely that some of the variability in response between different thromboplastin reagents may be attributable to differences in phospholipid compositions and/or ionic strength used in the manufacture of such reagents [13]. The different compositions of artificially depleted plasmas and the different sensitivities of thromboplastin reagents to individual clotting factors may explain why artificially depleted plasmas are not necessarily commutable with native plasmas in the PT/INR test.

Table 1.  Mean clotting factor levels (± standard deviation) and mean International Normalized Ratio (INR) of various plasma samples reported in the literature
FactorSamples from patients treated with vitamin K antagonistsLyophilized artificially depleted plasmas
Bertina [7]Kumar et al. [9]Lind et al. [10]Poller et al. [11]
  1. The levels reported by Bertina [6] were given in units mL−1, and were transformed to percentages, assuming 1 u mL−1 = 100%. The INRs reported by Poller et al. [10] were determined with a human reference thromboplastin (BCT) and with a rabbit reference thromboplastin (CRM149R).

FII (%)23 ± 41920.0 ± 9.711.4 ± 4.3
FVII (%)36 ± 113329.3 ± 1310.9 ± 3.6
FX (%)20 ± 41812.0 ± 4.921.5 ± 6.8
INR3.002.82.683.03 (BCT) or 3.46 (CRM149)

Poller et al. stated in their letter that I repeated data previously presented to a European Action on Anticoagulation (EAA) Annual Meeting in February 2006. In fact, the original measurements were performed after February 2006, and I reported the data in an oral presentation to the final meeting of EAA participants on 15–16 September 2007. This was a private meeting, and my data were not published by the EAA. The data were obtained in my laboratory with a single batch of rabbit thromboplastin PT Fibrinogen HS PLUS on a single ACL-300 coagulometer. Even if my coagulometer varied from other coagulometers, the data are still valid, because the clotting times of the lyophilized artificially depleted ECAA plasmas and the fresh native plasmas were determined with the same reagent and the same coagulometer. The Subcommittee on Control of Anticoagulation Guidelines does not require validation of certified plasmas by more than one laboratory [1]. The validation is essentially a comparison of fresh native plasmas’ INRs obtained via two different routes: the international standard route and the certified plasma route. My data showed that there was a discrepancy between the two routes, owing to non-commutability of all ECAA abnormal lyophilized plasmas for the particular rabbit thromboplastin reagent.

Discrepancies in INR with lyophilized samples have also been observed for other rabbit thromboplastins, and may be explained by various causes, e.g. additives and/or preservatives added to the plasmas before lyophilization or the process of lyophilization itself [14].

I would like to encourage the manufacturers or suppliers of ECAA calibrant plasmas to validate their materials in accordance with the Subcommittee on Control of Anticoagulation Guidelines [1], using fresh patient plasmas, and to report their reagent-specific validation data to the users of the ECAA plasmas.

Disclosure of Conflict of Interests

The author states that he has no conflict of interest.