1. NEQAS Survey 212 Factor IX 15/24
The importance of using an acceptable linear calibration curve for factor assays
Elaine Bromidge, Clare Dunsmore, Gary W. Moore
Viapath Analytics, Diagnostic Haemostasis & Thrombosis, Guy’s & St. Thomas’ NHS Foundation Trust, London, United Kingdom
Introduction
Generation and scrutiny of parallel line bioassay data are crucial to determine accurate factor assays1. At least three dilutions of test plasma are needed to determine linearity and parallelism. The coagulation data must be checked by looking at the slope value R2 and the shape of the curve. By using the parallel line method to determine relative factor potency this may show evidence of inhibition (most commonly the presence of antibodies to coagulation factors or lupus anticoagulants). A point to point curve or a single point curve can give a misleading result and inhibitors can be missed.
Observations
Analyses were carried out on the Sysmex CA-1500 and CS-2100i using Siemens Factor deficient plasma and SHP calibrator and Actin FS.
For the sample displayed in Figure 1, if single point analysis had been used, the above would have been underestimated at the 1/1 dilution equivalent to 24.8iu/dL, with no inhibitor effect observed.
The importance of using linear curves has been highlighted in NEQAS sample (Table 1). Results achieved at St Thomas’ were (1/1), (1-2) and (1/4) (mean = 121.6u/dL). The discrepancy across the dilutions revealed a loss of linearity at the top of the curve. Close scrutiny of the associated calibration curve showed that the assay was no longer linear below approximately 15% Factor IX activity2 and therefore the resulting curve skewed when forced into linear interpolation, emphasizing the importance of scrutiny of dose response data.
*NEQAS sample 15/24 was reanalysed using the updated Factor IX protocol described below and the results are shown in Table 1.
Figure 1: Test samples containing inhibitory activity show an apparent increase in relative potency as the samples is diluted (Sysmex CA-1500).
Original Protocol
Updated Protocol
Sysmex UK Calibration - Linear
Raw Data - Secs
1/2
53.0
2/1
53.9
1/4
59.6
3/2
56.2
1/8
66.6
1/1 60
1/16
73.4
66.5
1/32
79.4
79.7
Initial sample dilution 1/5
85.1
Low Curve
72.7
79.6
1/50
82.6
1/95
87.3
Initial sample dilution 1/20 and 1/5 for low curve
NEQAS Survey 212 Factor IX 15/24
NEQAS Median
106 u/dL
Original Result (mean)
121.6 u/dL
Grade D
Updated Result * (mean)
104.8 u/dL
Grade A
Table 1: NEQAS results
Table 2: Protocol amendments to the Factor IX assay.
Figure 2: Factor IX calibration curves on old and new protocols. Green = new protocol, blue = original protocol
Calibration curve protocol amendments & Outcomes
An evaluation was carried out by Sysmex and Viapath Analytics and it was determined that the CS-2100i protocol for Factor IX assays should be adjusted, as detailed in Table 2. The initial sample dilution was increased from 1/5 to 1/20 and additional dilutions were added to the standard curve and in consideration of the reduction in linearity at lower concentrations; a low curve was introduced to cover factor IX concentrations of less than 10%.
The multi dilution approach was kept in order to maintain the ability to observe potential presence of inhibitors across a range of dilutions whilst maintaining accuracy.
Each batch of Factor IX deficient plasma has dose response analysis as part of the batch acceptance process and the data are closely scrutinized to assess the limits of the central linear component and alter standard curve dilutions as appropriate to maximise linearity.
As a consequence of this investigation UK NEQAS now request the lot number detail of the reference and deficient plasma used as well as the sample dilutions used to generate the result.
Acknowledgements
The authors would like to acknowledge the support and advice of Ian Holding at Sysmex UK and UK NEQAS.
References
I Mackie, P Cooper, A Lariw, S Kitchen, E Gray, M Laffan ‘Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis(2012) British Committee for Standards in Haematology
R Mellick Factor IX investigation ( ) Document reference: SUKBMS Sysmex UK.