Evaluation of APTT clot waveform analysis by IL ACL TOP® in patients with Haemophilia and comparison to clot waveform analysis with MDA® 180 P-TH-568 Aghighi S., Riddell A.F., Vijayakumar, E., Chowdary, P. Katharine Dormandy Haemophilia Centre & Thrombosis Unit, The Royal Free Hospital, Pond Street, London NW3 2QG, U.K. Introduction Optical data generated during clotting tests such as prothrombin time (PT) and activated partial thromboplastin time (APTT) have been used to measure kinetics of fibrin generation, and indirectly of thrombin generation in haemophilia patients 1.   Formation of fibrin leads to a concomitant increase in the optical density of the plasma specimen (or a decrease in light transmittance). Modern automated photo-optical coagulation analysers, collect optical data over the entire course of clot formation and an optical profile, or clot waveform, is mathematically processed by a software algorithm, to derive parameters of coagulation velocity. Clot waveform analysis using light transmittance on MDA® 180 analysers has previously been reported 1,2,3. Recently, Solano et al. have studied the use of APTT derivative curves by using absorbance values to create the clot reaction curve on ACL TOP® analysers Instrumentation Laboratory (IL), USA) in different patient groups 4. Results Min1 correlated significantly with Max1 (r = 0.883, p < 0.0001) Min2 also correlated significantly with Max2 (r = 0.883, p < 0.0001) FVIII levels correlated significantly with Min1, Min2, Max1, and Max2 (r = 0.804, 0.837, 0.701, 0.75;respectivly; p < 0.0001) FIX levels were also correlated significantly with Min1, Min2, Max1, and Max2 (r = 0.721, 0.697, 0.685, 0.681 respectively; p < 0.001) Sensitivity and specificity of tests to distinguish between normal individuals and patients with Haemophilia A or B was Min1 = 99%, 96%, Min2 = 97%, 96%, Max1 = 98%, 96% and Max2 = 100%, 96%. Figure A Figure B Aim To investigate the use of clot reaction curve derivatives, based on light absorbance on the ACL TOP® coagulometer, and compare them with the light transmittance generated clot waveform as performed on the MDA® 180 analyser, in a normal individual group and in patients with haemophilia A. Methods APTTs were measured on the MDA® 180 (Organon Teknika, UK), using Platelin LS reagent (Stago, France) and on an ACL TOP® , using SynthASIL reagent (IL, USA). MDA® 180 clot waveform parameters (Min1 = velocity, Min2 = acceleration) and ACL TOP® clot profile parameters (Max1 = velocity, Max2 = acceleration) were calculated by the manufacturer’s software. A reference range was established for each parameter and compared with 70 patients with Haemophilia (Haemophilia A: severe, n=27, moderate n=9, mild n=10, and Haemophilia B: severe n=9, moderate n=3, mild n=4). FVIII and FIX activity levels were also measured with one-stage clotting factor assay using the ACL® 3000 analyser. Min2 % Min1 % Figure B showing linear regression analysis of ACL TOP® Max2 and MDA ® 180 Min2 (r2 = 0.789). Figure A showing linear regression analysis of comparison of ACL TOP® Max1 and MDA ® 180 Min1 (r2 = 0.771). Discussion Clot waveform analysis, based on light transmission on the MDA® 180 analyser, has been used to analyse fibrin clot formation beyond clot detection as a surrogate marker of thrombin generation. Several groups have described the usefulness of clot waveform analysis in different clinical settings 1,2,3. In our study light absorbance based APTT derivatives generated by the ACL TOP®, correlated significantly with parameters of clot waveform by the MDA® 180. The high sensitivity and specificity we achieved make them a useful global screening test. Our results showed a significant correlation between FVIII/FIX clotting activity levels and Max1/Max2. APTT velocity and acceleration parameters of fibrin polymerisation may not only be dependent on clotting factor levels but also other markers that may influence the coagulation potential of individuals. Further studies are required to determine the correlation of APTT derivatives and clinical phenotype of haemophilia patients. d e Max2 mAbs Max1 c a b Time (s) APTT clot reaction curve by ACL TOP®. Based on light absorbance, point a is the start of light absorbance by addition of activating reagent. Point b indicates the initiation of coagulation. Point c is the coagulation midpoint. Point d is the end of coagulation phase. Point e is the end of acquisition time. The absolute value of Max1 shows the maximum coagulation velocity and the absolute value of Max2 shows the maximum coagulation acceleration 4. APTT clot reaction curve by MDA® 180. Based on light transmittance, point a is the start of light transmittance by addition of activating reagent. Point b indicates the initiation of coagulation. Point c is the coagulation midpoint. Point d is the end of coagulation phase. Point e is the end of acquisition time. The absolute value of Min1 shows the maximum coagulation velocity and the absolute value of Min2 shows the maximum coagulation acceleration 1. Conclusion The APTT clot reaction derivatives, produced by the ACL TOP® correlated significantly with the parameters of clot waveform analysis on the MDA® 180. Similar to MDA clot waveform analysis, ACL TOP® results showed a significant correlation with FVIII/FIX levels. The high sensitivity and specificity of these tests make them a reliable predictor of FVIII/FIX levels in haemophilia patients. References Braun, P.J., Givens, T.B., Stead, A.G., Beck, L.R., Gooch, S.A., Fischer, T.J. (1997). Properties of optical data from APTT and PT assays. Thromb Haemost, 78, 1079-87. Downey, C., Kasmir, R.. Toh, CH. (1997). Novel and diagnostically applicable information from optical waveform analysis of blood coagulation in disseminated intravascular coagulation. British Journal of Haematology, 98(1), 68-73. Shima, M., Matsumoto, T., Fukuda, K., Kubota, Y., Tanaka, I., Nishiya, K (2002). The utility of activated partial thromboplastin time (aptt) clot waveform analysis in the investigation of hemophilia a patients with very low levels of factor viii activity (FVIII:C). Thromb Haemost, 87(3), 436-441. Solano, C., Zerafa, P., Bird, R. (2010). A study of atypical APTT derivative curves on the ACL TOP coagulation analyser. Int Jnl Lab Hem 33(1), 67-78.