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.
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
R² = 0.9998
100
10
Figure 1: Test samples containing inhibitory activity show an
apparent increase in relative potency as the samples is diluted
(Sysmex CA-1500).
OD
1
Original Protocol
Sysmex UK
Raw Data Calibration - Linear Secs
1/2
53.0
1/4
59.6
1/8
66.6
1/16
73.4
1/32
79.4
Initial sample dilution 1/5
Table 2: Protocol
amendments to the Factor
IX assay.
Updated Protocol
Sysmex UK
Raw Data Calibration - Linear Secs
2/1
53.9
3/2
56.2
1/1
60
1/2
66.5
1/4
73.4
1/8
79.7
1/16
85.1
1/16
72.7
1/32
79.6
1/50
82.6
1/95
87.3
Low Curve
*NEQAS sample 15/24 was reanalysed using the updated Factor IX
protocol described below and the results are shown in Table 1.
1000
Concentration (MPL)
The importance of using linear curves has been highlighted in NEQAS
sample 15.24 (Table 1). Results achieved at St Thomas’ were 117.3
(1/1), 122.2 (1-2) and 125.3 (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.
Standard Curve for IgM Assay
Initial sample dilution 1/20 and
1/5 for low curve
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
1. 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
2. R Mellick Factor IX investigation (12.06.2015) Document reference: SUKBMS13-411 Sysmex UK.