Background: The differentiation of coagulation disorders with prolonged aPTT is important for appropriate treatment. Mixing test is useful for screening coagulation factor deficiencies, inhibitors, and lupus anticoagulants (LA), but this assay is a complicated work and requires 2-hour incubation. In addition, interpretation of the results is sometimes difficult.

Aims: To differentiate factor deficiencies, inhibitors, and LA-positive cases by a short-time incubation and the numerical parameter index using aPTT-based clot waveform analysis (CWA).

Methods: aPTT-CWA was performed with CP3000^™ apparatus using Coagpia APTT-N^®. FVIII-deficiency (n=20), LA-positive (n=6), and FVIII inhibitor plasma (n=48; 0.6-302 BU/mL) from patients and commercial plasma were used. Pooled normal plasma (PNP) and samples were mixed (1:1) and aPTT waveform were obtained immediately (0 min-incubation) and after 12 min-incubation. The differentiation was performed in 2 steps. To first differentiate FVIII inhibitor, ratio of 12 min/0 min of 4 parameters (clotting time, min1, min2, Aspect ratio (Ar:presentated in ISTH2019, ID PB1302) were assessed. Following, to differentiate LA and FVIII-deficiency, the above parameters were assessed.

Results: In CWA with FVIII inhibitor sample, the clotting time ratio was increased and the other ratios were decreased significantly (p< 0.01) compared to the ratios in factor-deficiency and LA-positive (Figure 1). By setting the different mixing ratios including further 2 patterns (sample:PNP = 9:1, 1:1, 1:9), the sensitivity of differentiation in inhibitor with lower (< 2 BU) and higher titer (>100 BU) was improved, although only clotting time ratio in cases with lower inhibitor titer showed no significant difference. The 4 parameters between LA-positive and factor-deficiency cases showed significant differences (p< 0.01, Figure 2).

Conclusions: We successfully established a novel mixing test based on aPTT-CWA to precisely differentiate FVIII-deficiency, FVIII inhibitors, and LA-positive in a short-time incubation. This method will greatly contribute prompt diagnosis of prolonged aPTT samples.

[Figure 1. ratio of 4 parameters (clotting time, min1, min2, Ar). * : p<0.01, ** : p<0.001]

[Figure 2 . Parameters for differentiation between LA and Haemophilia A (HA)]

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