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. 2000 Jun;53(6):470–475. doi: 10.1136/jcp.53.6.470

Photodynamic treatment of pooled coumarin plasma for external quality assessment of the prothrombin time

A M H P van den Besselaar 1, A Moor 1
PMCID: PMC1731213  PMID: 10911807

Abstract

Aims—To determine the conditions of photodynamic inactivation of vesicular stomatitis virus (VSV) added to pooled coumarin plasma and the effects of the photodynamic treatment on the prothrombin times and international normalised ratio (INR) in a Netherlands national external quality assessment scheme.

Methods—Pooled coumarin plasma samples were illuminated with visible light in the presence of 1 µM methylene blue. Inactivation conditions for VSV in pooled coumarin plasma were determined using an end point dilution assay. Plasma illuminated for 20 minutes was mixed with red blood cells and mailed to participants of the Netherlands external quality assessment (EQA) scheme. Prothrombin times and INRs were determined with various thromboplastin reagents.

Results—Photodynamic treatment using 1 µM methylene blue and 700 W/m2 caused 4.7 log inactivation of VSV in pooled coumarin plasma. Fibrinogen and coagulation factors II, V, VII, and X were decreased slightly by the treatment. These conditions caused prolongation of the prothrombin time in EQA surveys. The magnitude of the effect was different for various thromboplastin reagents. The increase of the INR was negligible when measured with the Thrombotest reagent. With other reagents, an approximately 5–16% increase of the INR was observed. Interlaboratory variation of the INR was not affected by photodynamic treatment.

Conclusions—Photodynamic treatment of pooled coumarin plasma is very effective for the inactivation of some enveloped viruses such as VSV, but has only a limited effect on the prothrombin time and INR. Photodynamic treatment can be used to improve the viral safety of coumarin plasma for EQA of the prothrombin time and INR.

Key Words: prothrombin time • international normalised ratio • external quality assessment • photodynamic treatment

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Figure 1 Thrombotest dilution plots of normal plasma (cross), pooled coumarin plasma before illumination (open circle) and after illumination with methylene blue for 20 minutes (open square). The regression equations for the latter two plots are y = 47.3 + 27.6x and y = 48.0 + 30.1x, respectively. The intercept of the line for normal plasma with the Y axis is named tmin, uninh.. I is the distance on a horizontal line between tmin, uninh. and the intercept of the line for coumarin plasma with that horizontal line.

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Figure 2 Infectivity of vesicular stomatitis virus (VSV) after treatment with 1 µM methylene blue and incandescent light (700 W/m2). The VSV titer is expressed on a log scale. Two different pooled coumarin plasmas were used in two separate experiments (open squares and circles).

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Figure 3 Fibrinogen activity of pooled coumarin plasma after treatment with 1 µM methylene blue and incandescent light (700 W/m2). Before illumination, either air (open circle) or nitrogen (open square) was bubbled through the plasma, or no additional treatment was given (cross).

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Figure 4 Coagulation factor activities of pooled coumarin plasma after treatment with 1 µM methylene blue and incandescent light (700 W/m2). Factor V activity was measured in plasma treated with air (open circle), or nitrogen (open square). In plasma without preceding air or nitrogen treatment, factor V (cross), factor II (closed circle), factor VII (closed triangle), and factor X (closed square) were measured.

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Figure 5 International normalised ratio (INR) of pooled coumarin plasma determined with recombinant human thromboplastin (Innovin) after treatment with 1 µM methylene blue and incandescent light (700 W/m2). Before illumination, either air (open circle) or nitrogen (open square) was bubbled through the plasma, or no additional treatment was given (cross).

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Figure 6 International normalised ratio (INR) of pooled coumarin plasma determined with bovine thromboplastin (Thrombotest) after treatment with 1 µM methylene blue and incandescent light (700 W/m2). Before illumination, either air (open circle) or nitrogen (open square) was bubbled through the plasma, or no additional treatment was given (cross).

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