A monitor system is defined as a combination of a brand of monitor with a single lot of test strips/cards. Two different test systems (CoaguChek Mini and TAS PT-NC) were loaned to each of the 10 ECAA centres by Roche Diagnostics (Mannheim, Germany) and Bayer AG (Leverkusen, Germany), respectively.
The CoaguChek system used lot 164 “Mini” test strips, incorporating rabbit thromboplastin.
The TAS monitor, recently redesignated the RapidPointCoag, was used with “PT-NC” test cards (lot 307060002), containing human placental thromboplastin.
Both test systems have been described in detail in previous ECAA reports.3–5
To perform a reliable ISI calibration, real PTs (in seconds) are required.6
Therefore, a master code chip to replace the conventional code chip was provided by the manufacturer of the CoaguChek Mini system to display real PT values. In the case of the TAS PT-NC, a specific correction formula was supplied to us by the manufacturer to restore the monitor displayed PT to the real PT value.
IRPs for thromboplastin
The World Health Organisation (WHO) human recombinant (rTF/95)7
and the rabbit plain (RBT/90)8
IRPs were provided by the WHO for same species ISI calibrations because the CoaguChek Mini and TAS PT-NC test systems use rabbit and human based thromboplastin, respectively.
Scientific assistants performing the exercise at the 10 ECAA centres attended a training workshop to familiarise them with the two types of monitor and to standardise the manual tilt tube technique.
Quality control exercise
A preliminary quality control exercise consisted of the testing of five abnormal lyophilised plasma samples prepared at the central facility. The purpose was to detect any monitor system or operator consistently providing outlying results. Results from all centres were satisfactory.
Whole blood and fresh plasma samples
The collection and testing of non-citrated whole blood on the monitors and the collection of fresh plasma samples for manual PT testing with the IRP were described in detail in a previous ECAA report.4
All donors gave informed consent.
Lyophilised plasma samples
Abnormal lyophilised plasma samples (artificially depleted, individual coumarin, pooled coumarin) and lyophilised normal plasma samples were prepared at the ECAA central facility.
Artificially depleted plasma samples
Sets of 60 artificially depleted lyophilised plasma samples were prepared using ECAA methodology9
from plasmapheresis donations. They were selected to span the INR range 1.5–4.5 with ECAA rabbit reference thromboplastin, a secondary reference thromboplastin.10
All the lyophilised, artificially depleted plasma samples had a fibrinogen content greater than 1.5 g/litre and factor V values greater than 50%, with inter-vial coefficient of variation (CV) values of less than 3%.
Individual coumarin plasma samples
Sets of 60 lyophilised coumarin plasma samples were prepared. Venous blood samples (20 ml) were collected from 60 adult patients stabilised on long term warfarin treatment in the anticoagulant clinic, Manchester Royal Infirmary, UK. For ethical reasons, the volume of blood collected from each patient had to be limited and therefore was insufficient for inter-vial CV measurements.
Pooled coumarin plasma samples
These were collected at Leiden University Medical Centre, The Netherlands, into 10 separate pools from 75–80 patients stabilised on long term oral anticoagulant treatment. The samples spanned the INR range 1.5–4.5, with an inter-vial CV of less than 3%.
Normal plasma samples
Plasma samples, collected from 20 healthy adult volunteers, were lyophilised by the ECAA central facility. The donations had to be limited to 20 ml of whole blood, so the volume was insufficient for inter-vial studies.
Reconstitution and testing of lyophilised plasmas on POCT PT monitors
Each lyophilised plasma sample was restored to its original volume of 0.5 ml with distilled water and left at room temperature for at least 10 minutes before testing. Plasma samples were tested within 30 minutes of reconstitution.
Reconstituted plasma (0.1 ml) was transferred to a plastic tube, 0.1 ml of 17mM calcium chloride was added, and the sample was mixed. The recalcified plasma was applied within five seconds to the test strip/cards and the displayed monitor PT recorded. The calcium chloride was provided to all centres by the ECAA central facility.
The 10 ECAA laboratories tested all lyophilised plasma samples on the monitors using single tests because of the restricted number of test strips/cards of the same individual lots. The 60 artificially depleted plasma samples, 60 individual coumarin plasma samples, 10 pooled coumarin plasma samples, and 20 normal plasma samples were tested on both POCT monitor systems using the same lots of test strips/cards at all centres.
Certification of lyophilised plasma samples
Three centres (Leiden, Manchester, and Milan) provided the certified PT values for the lyophilised plasmas by duplicate tests with both WHO rabbit (RBT/90) and human (rTF/95) IRPs using the manual technique. Certified PTs were the geometric means of the duplicate PTs obtained with the relevant IRP at the three centres. The certified INR values were obtained using the certified PT, the mean normal prothrombin time (MNPT) derived from the lyophilised normal samples, and the stated ISI of the IRP.
The ISI for whole blood and lyophilised plasma calibrations with the two monitor systems were derived by the recommended WHO orthogonal regression procedure.6,11
Results with coumarin or artificially depleted plasma samples were excluded if their INR values, calculated with the relevant IRP, were outside the INR range 1.5–4.5. For the calibrations with whole blood, individual normal and abnormal log PTs obtained with the IRP were plotted, in the customary way, on the vertical axis against the log PT obtained from the monitor system on the horizontal axis, and the slope of the calibration line was calculated using orthogonal regression analysis. For the calibrations using lyophilised plasma samples, certified log PTs were plotted on the vertical axis in place of the log PT results obtained with the manual technique and the thromboplastin IRP in conventional ISI calibrations.
The SD of the log PT on the monitor and with the IRP was estimated (defined as “residual SD” in the WHO guidelines),11
and samples were excluded if the perpendicular distance from the orthogonal regression line was greater than three times the residual SD. The final orthogonal regression line was determined from the remaining data. The precision of the calibration slope (b
) was measured by its CV, CV(b
) = 100 × SE(b
, where SE(b
) is the standard error of b
The ISI was derived as ISI = b
is the ISI of the IRP. Between centre ISI variation was measured using the CV (%). Paired t
tests were performed to compare the mean ISI derived from whole blood calibrations with the mean ISI derived from the three types of lyophilised plasma calibrations. Outlying ISI values were detected using an algorithm described and used by van den Besselaar8
in the calibration of the WHO rabbit plain IRP (RBT/90) and also used by Tripodi and colleagues7
in their calibration of the WHO human plain IRP (rTF/95).
Calibrations with the 60 lyophilised artificially depleted and 60 individual coumarin plasma samples incorporated the 20 normal plasma samples. Because only 10 pooled coumarin plasma samples were available, the number of normal plasma samples was reduced to three to preserve the proportion of normals to abnormals recommended by the WHO guidelines.11
The first three normal plasma samples were selected arbitrarily at each centre for inclusion.
INR comparison for target patient samples: reliability of INR
The relative reliability of lyophilised plasma ISI calibration of whole blood POCT monitors was assessed from the 600 fresh coumarin samples collected at the 10 centres. Five hundred and thirty five of these (target samples) had INR values within the 1.5–4.5 INR range with both the IRPs. For each target sample, monitor INR (INRm) were determined using the whole blood sample PT (PTWB) and the individual centre MNPT of whole blood (MNPTWB), with the respective individual centre ISI derived from the lyophilised plasma calibration (ISILY); that is:
Monitor displayed INR values were compared with the reference (“true”) INR (INRr) obtained on the same target samples at the same centre using the manual PT test with the species specific WHO thromboplastin IRP.
INR differences were plotted against the mean of the monitor and “true” INR12
to assess whether the INR differences were related to the intensity of anticoagulation. Because INR differences increased as the average INR increased, log INR were analysed, as recommended by Bland and Altman,12
and geometric means of the monitor and reference INR were calculated. The mean INRs were compared using paired t
tests and confidence intervals.