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A modified chromogenic assay for the measurement of very low levels of factor VIII activity (FVIII:C

Summary.  A precise and sensitive chromogenic assay for the measurement of very low levels of factor VIII (FVIII) in plasma has been developed. The assay is based on modifications of a commercially available chromogenic assay. The modifications
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  A modified chromogenic assay for the measurement of verylow levels of factor VIII activity (FVIII:C) R. YATUV, I. DAYAN and M. BARU Omri Laboratories Ltd, Weizmann Science Park, Nes-Ziona, Rehovot, Israel  Summary.  A precise and sensitive chromogenic assayfor the measurement of very low levels of factor VIII(FVIII) in plasma has been developed. The assay isbased on modifications of a commercially availablechromogenic assay. The modifications include reduc-tionofsamplefinaldilutionfactorandprolongationof the development period. The modified assay allowsaccurate and precise measurement of FVIII in therange of 0.001–0.02 IU mL ) 1 . The detection limit is0.0005 IU mL ) 1 and the quantitation limit is0.0015 IU mL ) 1 . This assay can be used in researchand to study the clinical efficacy of low circulatinglevels of FVIII in haemophilia A patients. Keywords : chromogenic assay, factor VIII, haemo-philia A Introduction Accurate determination of the level of procoagulantfactor VIII (FVIII:C) is necessary in order to assessthe severity of the defect in haemophilia A patientsand to correlate clinical phenotype in circulatingFVIII:C levels. The methods to measure FVIII levelsinclude clotting methods [1], chromogenic assay [2],thrombin generation assay [3,4] and activated partialthromboplastin time clot waveform analysis [5].However, the quantitation limit and precision of these methods are limited at FVIII levels of <0.01 IU mL ) 1 . [6]. Therefore, there is a scientificand clinical need to develop a simple and accuratemethod that can detect and quantify very low levelsof FVIII in plasma of haemophilic patients.The chromogenic method for determination of FVIII activity was introduced more than 2 decadesago[2].ThemethodisbasedontheadditionofdilutedFVIII samples to a mixture of reagents and factor IXa(FIXa), which results in the formation of factor Xa(FXa). FXa then hydrolyses a chromogenic substrateliberating a chromogenic group. The intensity of theproduced colour is proportional to FVIII concentra-tion. Several different chromogenic kits are commer-ciallyavailable,buttheirdetectionlevel(DL)islimitedto0.005 IU mL ) 1 andtheirreliabilityandprecisionatthislevelare nothigh[6].TheCoamaticchromogenicassay kit (Chromogenix, Milan, Italy) measures FVIIIcofactor activity based on FX activation to FXa byFIXa, in the presence of Ca ++ ions, phospholipids andthrombin. FXa hydrolyses the chromogenic substrateS-2765, liberating the chromogenic group  p -nitroan-iline (pNA) and producing a yellow colour able to bereadphotometricallyat405 nm.Thecolourproducedis linearly related to the amount of FVIII in the testsample, with a detection limit of 0.005 IU mL ) 1 forFVIII using the Coamatic assay.In this study, we modified the Coamatic assay andshowed that sensitivity of the assay can be highlyenhanced. The modifications included: (i) reductionin the final dilution factor of the samples to 1:30(instead of 1:80); and (ii) prolongation of thedevelopment step, the incubation period with thesubstrate, to 30 min (instead of 10 min). Thesemodifications enabled the determination of FVIII ata DL of 0.0005 IU mL ) 1 and a quantitation level(QL) of 0.0015 IU mL ) 1 . As this assay is simple andmore sensitive than any other known method fordetection of low levels of FVIII, it can be used inresearch and to study the clinical efficacy of lowcirculating levels of FVIII in haemophilia A patients. Materials and methods Standard and FVIII-deficient plasma An international standard of FVIII concentrate wasobtained from the National Institute for Biological Correspondence: Moshe Baru, DSc, Omri Laboratories, Bldg 22,Weizmann Science Park, Nes-Ziona, PO Box 619, Rehovot 76106,Israel.Tel.: +972 8 9302970; fax: +972 8 9302975;e-mail: after revision 9 January 2006 Haemophilia  (2006),  12,  253–257 DOI: 10.1111/j.1365-2516.2006.01209.x   2006 Blackwell Publishing Ltd 253  Standards and Control (NIBSC, Hertfordshire, UK).The standard is the Seventh International Standardfor Blood Coagulation FVIII:C, containing freeze-dried concentrate of plasma-derived FVIII, formula-ted with human albumin. The preparation wasestablished by the Expert Committee on BiologicalStandardization of the World Health Organization inNovember 2003.Pooled citrated plasma of severe haemophiliapatients (SHP) was obtained from HRF Inc. (Raleigh,NC, USA). Original Coamatic  assay Coamatic  FVIII kit (Chromogenix, Milan, Italy)components were reconstituted and prewarmed,according to the manufacturer’s instructions. Briefly,working solutions were as follows: (i) assay buffer25 m m  Tris pH 7.9, 0.9% NaCl, 1% BSA; (ii) factorreagent containing bovine FIXa (0.05 U mL ) 1 ),FX (0.45 IU mL ) 1 ) and thrombin (0.17 NIH-U)co-lyophilized with CaCl 2  (6.7 m m ) and phospholi-pid (0.03 m m ); and (iii) S-2765 (Xa substrate,0.43 mg mL ) 1 ) with I-2581 (thrombin inhibitor0.011 mg mL ) 1 ).A standard of FVIII concentrate was reconstitutedin distilled water according to the manufacturer’sinstructions and diluted to 0.1 IU mL ) 1 in SHPplasma. The diluted FVIII standard was furtherdiluted in Coamatic kit buffer in a two-step proce-dure: a predilution to prepare 0 (buffer blank),0.001, 0.0025, 0.005, 0.0075, 0.01, 0.015 and0.02 IU mL ) 1 standard dilution set, and a finaldilution of 1:80 of all samples by the addition of 12.5  l L of sample to 1 mL of assay buffer. Allcomponents were added to microtitre plates usingEppendorf pipettes with matching dispenser tips.Samples (50  l L of each in triplicate) were loadedin a microplate wells and warmed for 4 min at37   C. Prewarmed factor reagent (50  l L per well)was added and the plate was mixed gently andincubated for an additional 4 min at 37   C. Pre-warmed S-2765 substrate (50  l L per well) wasadded and the plate was mixed gently and incubatedfor an additional 10 min at 37   C. The reaction wasstopped by addition of 20% acetic acid (50  l L perwell), the plate was shaken gently and the absorb-ance at 405 nm for pNA chromogenic group wasread photometrically in each well by VERSAmaxtunable microplate reader (Molecular Devices, Sun-nyvale, CA, USA). The development period for thisassay method is the period of time between theaddition of the S-2765 substrate and the stopping of the assay by acetic acid addition. Modified Coamatic  assay The assay was performed similarly to the srcinalmethod with modifications of two of the assayparameters: (i) 3 series of final dilutions (1:20, 1:30and 1:50) were prepared by addition of 50, 33 or20  l L of the prediluted sample to 1 mL of assaybuffer respectively; the samples were tested in com-parison with the recommended 1:80 final dilution;and (ii) the development period was extended to 20,30, 40, 50 or 60 min, in comparison with therecommended 10 min. In both the original andmodified Coamatic tests, optical density (OD) valueswere plotted vs. FVIII concentrations, arithmetically. Statistical evaluation Microsoft Excel Program, version 2002 SP3, Micro-soft Corp. (Redmond, WA, USA). GraphPad PrismProgram, version 4.02, GraphPad Software Inc. (SanDiego, CA, USA) were used. The DL and the QL of the assay methods were calculated according to theICH Guidelines, Q2B, Validation of AnalyticalProcedures: Methodology [7]. In summary, a stand-ard curve was prepared and the slope of the linearregression line was determined. Ten blank sampleswere evaluated and the standard deviation of the 10responses was calculated. The DL and QL for themethod were then calculated according to thefollowing formulae:DL ¼ 3 : 3  standard deviation of blankSlope of regression line  ; QL ¼ 10  standard deviation of blankSlope of regression line  : Results The effect of final-dilution factor on assay sensitivity In order to develop a method for measuring verylow levels of FVIII an accurate standard curve hasto be evaluated. Therefore, we used calibrated FVIIIconcentrate (seventh standard 99/678 from theNIBSC) to generate a reliable calibration curveand a modified chromogenic assay was used tomeasure FVIII activity. Four sets of calibrationcurves (0.001–0.02 IU mL ) 1 ) were tested by theCoamatic chromogenic assay according the manu-facturer’s instructions, except that the final dilutionswere 1:20, 1:30, 1:50 and 1:80 (1:80 is the dilutionrecommended by the manufacturer). After thedevelopment period of 30 min, the absorbance at 254  R. YATUV  et al. Haemophilia  (2006),  12 , 253–257    2006 Blackwell Publishing Ltd  405 nm was measured by ELISA reader and theresults are presented in Fig. 1.Analysis of OD values indicates that linearity wasbest fitted with a final dilution of 1:30 ( R 2 ¼  0.981).The slope of the standard curves increased as thedilution factor decreased, enabling one to betterdistinguish between adjacent values on the curve.Since the 1:30 dilution gave the best fitted linearity(>0.98) with a relatively large slope, it was chosenfor further analysis. The effect of development period on assay sensitivity Extension of the development period was tested for astandard curve with a final dilution of 1:30. Thesamples were incubated with the substrate forperiods of 10, 20, 30, 40, 50 or 60 min andabsorbance at 405 nm was measured by ELISAreader. The results presented in Fig. 2 indicate thatthe slope of the linear fitted curve increased with theincrease in development time, but the  R 2 values of the fitted linear line decreased to <0.98 at 40 to60 min incubation time. Therefore, an incubationtime of 30 min, which gave good fitted linearity( R 2 ¼  0.981) with significant slope value (33.9), wasselected as the preferred incubation time for thisassay. It is notable that the addition of 20% aceticacid to terminate the reaction does not changesignificantly the calibration curve and any of thecurve parameters (data not shown). Comparison between the srcinal and the modified  protocols After choosing the best assay modifications, a directcomparison between the modified (final dilution of 1:30, 30 min of development period) and unmodifiedprotocol (final dilution of 1:80, 10 min of develop-ment period) was performed. A standard curve wasprepared using calibrated FVIII concentrate (seventhstandard 99/678, NIBSC, UK) diluted in a buffer(1:80 or 1:30) and FVIII activity was measured. Theresults are presented in Fig. 3. Since a prolongeddevelopment time yielded elevated OD values of theblank, the blank values were subtracted from bothcurves to make the comparison easier. In addition,the DL and QL were calculated as described inMaterials and methods and are presented in Fig. 3.As can be seen in Fig. 3, the slope of the modifiedassay linear curve is five times higher than that of theunmodified curve, resulting in the DL and QL valuesof the unmodified protocol (0.0005 and 0.0015)being five times lower than those of the modifiedprotocol (0.0025 and 0.0076). In addition the R 2 value of the modified protocol (0.981) was higherthan that of the unmodified protocol (0.939). Measurement of low FVIII levels in plasma of severehaemophilic patients Measurement of low FVIII levels in plasma of severe haemophilic patients was evaluated by FVIII (IU mL –1 )    A   4   0   5  n  m R  2 SlopeInter.0.962 42.7 0.460.981 33.9 0.430.973 23.7 0.430.924 15.6 0.43 Fig. 1.  Effect of sample dilution. A standard dilution set (0.001–0.02 IU mL ) 1 ) of seventh international standard 99/678 factorVIII concentrate was analysed using a Coamatic chromogenicassay with final dilutions of 1:20 ( n ), 1:30 ( ), 1:50 ( ) or 1:80( ) and 30-min incubation with S-2765 substrate. Data aremean ± SD of three independent experiments.  R 2 , slope andintercept values of each curve are indicated. FVIII (IU mL –1 )    A   4   0   5  n  m  0.981 33.9 0.43 R  2 SlopeInter.0.940 44.1 0.76 0.958 42.4 0.66 0.974 39.1 0.54 0.981 25.7 0.32 0.984 15.2 0.21 Fig. 2.  Effect of development time. A standard dilution set (0.001–0.02 IU mL ) 1 ) of seventh international standard 99/678 factorVIII concentrate was analysed using a Coamatic chromogenic as-say with incubations of 10 ( ), 20 ( ) 30 ( ), 40 ( h ), 50 ( ) or 60( n ) min with S-2765 substrate. Data are mean ± SD of threeindependent experiments.  R 2 , slope and intercept values of eachcurve are indicated. MEASUREMENT OF VERY LOW LEVELS OF FVIII:C  255   2006 Blackwell Publishing Ltd  Haemophilia  (2006),  12 , 253–257  spiking low concentrations of FVIII into a pool of SHP plasma, thus mimicking plasma of haemophi-lia patients with very low FVIII levels. Theinternational standard FVIII concentrate 99/678was reconstituted in distilled water according tothe manufacturer’s instructions and diluted to0.1 IU mL ) 1 in SHP plasma. The diluted FVIIIstandard was spiked into SHP plasma or a bufferto prepare 0.001–0.02 IU mL ) 1 standard curves.All samples were diluted to a final dilution of 1:30and analysed according to the modified procedure.Results of three independent experiments are pre-sented in Fig. 4a (including the blank background)and Fig. 4b (following subtraction of buffer andplasma blank values). The results indicate that twoparallel curves were obtained with very similarslopes for FVIII in buffer (slope  ¼  34) and inplasma (slope  ¼  32.8) (Fig. 4a). The intercept var-ied between the two curves because of a differentbackground absorbance of buffer and plasma.When blank values of buffer and plasma weresubtracted from sample OD, values in the curveswere very similar (Fig. 4b) indicating that the assayis not affected by the presence of plasma compo-nents and that the assay can be used for monitor-ing very low levels of FVIII in plasma of severehaemophilia A patients. Discussion The need for accurate measurement of very lowlevels of FVIII has increased in the last few yearsto allow for better monitoring of haemophiliapatients on replacement therapy, development of long-acting FVIII products [8], and for the assess-ment of circulating FVIII levels in gene-therapyclinical trials. Patients with severe haemophilia A,having low FVIII levels of <0.01 IU mL ) 1 , may differconsiderably in their bleeding tendency [9] possiblydue to variations in FVIII activity below DL. A recentstudy of prophylactic treatment in patients withsevere haemophilia indicated that even levels of FVIIIor FIX below 0.01 IU mL ) 1 may be sufficient toprotect against spontaneous bleeding [10]. Recent (IU mL –1 )    A   4   0   5  n  m  R  2  – 0.981 Slope – 33.9 DL – 0.0005 QL – 0.0015 R  2  – 0.939 Slope – 6.6 DL – 0.0025 QL – 0.0076 Fig. 3.  Direct comparison of srcinal and modified protocol. Astandard dilution set (0.001–0.02 IU mL ) 1 ), of seventh interna-tional standard 99/678 factor VIII concentrate in a buffer wasanalysed using a Coamatic chromogenic assay with the srcinal( ) or the modified ( ) protocols. Data are mean ± SD of threeindependent experiments. Blank values (buffer only) were sub-tracted from both curves.  R 2 , slope, DL and QL values of eachcurve are indicated. (a)(b) 00.0050.010.0150.020.025 FVIII (IU mL –1 )    A   4   0   5  n  m R  2 SlopeInter.0.976 34.0 0.430.963 32.8 0.2600. FVIII (IU mL –1 )    A   4   0   5  n  m R  2 Slope0.976 34.0 0.963 32.8 Fig. 4.  Standard dose–response in plasma and buffer. A standarddilution set (0.001–0.02 IU mL ) 1 ), of seventh internationalstandard 99/678 factor VIII concentrate in a buffer ( ) and pool of plasma of haemophilic patients ( h ) were analysed using themodified protocol. Data of OD values (a) and after subtraction of blank values of buffer and plasma (b) are mean ± SD of threeindependent experiments.  R 2 , slope and intercept values of eachcurve are indicated. 256  R. YATUV  et al. Haemophilia  (2006),  12 , 253–257    2006 Blackwell Publishing Ltd  clinical trials of FVIII and FIX gene therapy showedclinical efficacies at very low factor levels: a study of non-viral FVIII gene transfer presented a clinicalimprovement (demonstrated by a decrease in thefrequency of spontaneous bleeding episodes and inthe use of exogenous FVIII) even though factor levelswere relatively low [11]. Another study showed thatafter adeno associated virus-mediated FIX genetransfer, the bleeding frequency of a treated patientwas significantly reduced although there was nosubstantial change in FIX levels measured withcurrent available chromogenic and clotting methods[12].Attempts have been made to determine the coagu-lation potential in the presence of low FVIII activityusing advanced technologies. These include throm-bin generation [3,4], rotational thromboelastography[8] and waveform analysis [5]. However, the corre-lation between test results and FVIII concentrationsare not always conclusive. In addition, these tech-nologies require specific equipment and qualifica-tions, which are not common in all laboratories.Chromogenic assays for determination of FVIIIactivity are reliable and a common practice in mostcoagulation laboratories. However, the commer-cially available chromogenic kits cannot quantifyFVIII at levels of <0.005 IU mL ) 1 . Therefore, weaimed to improve the sensitivity of a well-establishedcommercial kit so as to enable the detection andquantitation of FVIII at levels of <0.005 IU mL ) 1 .Two modifications were evaluated: a decrease insample final-dilution factor and a prolongation of thedevelopment step. Using this modified protocol, weanalysed a low range calibration curve of 0.001–0.02 IU mL ) 1 (Figs. 1 and 2). The modified methodproduces a linear standard curve with a relativelylarge slope. DL and QL obtained with the modifiedprotocol (0.0005 and 0.0015 IU mL ) 1 respectively)are significantly (five times) lower than those of theoriginal protocol (0.0025 and 0.0076 IU mL ) 1 respectively Fig. 3). Hence, the modified protocolenables the quantitation of FVIII concentrationsdown to 0.0015 IU mL ) 1 without affecting thesimplicity of the test and its suitability for automa-tion. Similar sensitivity was found in plasma of SHPspiked with low levels of FVIII (Fig. 4), indicatingthat there is no interference of plasma components inthe assay and that the assay can be used formonitoring very low levels of FVIII in plasma of severe haemophilia A patients. In summary, thedescribed assay is reliable, simple and more sensitivethan any other previously described method fordetection of low levels of FVIII. Therefore, it can beused in research and to study the clinical efficacy of low circulating levels of FVIII in haemophilia Apatients. References 1 Over J. Methodology of the one-stage assay of FVIII(FVIII:C).  Scand J Haematol   1984;  33 : 11–24.2 Rosen S, Andersson M, Blomba ¨ck M  et al.  Clinicalapplications of a chromogenic substrate method fordetermination of FVIII activity.  Thromb Haemost  1985;  54 : 811–23.3 McIntosh JH, Owens D, Lee CA, Raut S, BarrowcliffeTW. A modified thrombin generation test for themeasurement of factor VIII concentrates.  J ThrombHaemost   2003;  1 : 1005–11.4 Beltran-Miranda CP, Khan A, Jaloma-Cruz AR andLaffan MA. Thrombin generation and phenotypiccorrelation in haemophilia A.  Heamophilia  2005;  11 :321–34.5 Shima M, Matsumoto T, Fukuda K  et al  . The utility of activated partial thromboplastin time (aPTT) clotwaveform analysis in the investigation of hemophilia Apatients with very low levels of FVIII activity (FVIII:C). Thromb Haemost   2002;  87 : 431–41.6 Barrowcliffe TW. Monitoring haemophilia severity andtreatment: new or old laboratory tests.  Haemophilia 2004;  10 : 101–14.7 ICH.  International Conference on Harmonisation of Technical Requirements for Registration of Pharma-ceuticals for Human Use – ICH Harmonized TripartiteGuideline. Validation of Analytical Procedures: Meth-odology Q2B , 1996 ( Baru M, Carmel-Goren L, Barenholz Y  et al.  FactorVIII efficient and specific non-covalent binding toPEGylated liposomes enables prolongation of itscirculation time and haemostatic activity.  ThrombHaemost   2005;  93 : 1061–8.9 Ingerslev J, Poulsen LH and Sorensen B. Potential roleof the dynamic properties of whole blood coagulationin assessment of dosage requirements in haemophilia. Haemophilia  2003;  9 : 341–52.10 Petrini P. What factors should influence the dosage andinterval of prophylactic treatment in patients withsevere haemophilia A or B.  Haemophilia  2001;  7 : 91–102.11 Roth DA, Tawa NMD, O’Brien JM, Treco DA andSelden R. Nonviral transfer of the gene encoding factorVIII in patients with sever hemophilia A.  N Engl J Med  2001;  344 : 1731–4212 Manno CS, Chew AJ, Larson PJ  et al  . AAV mediatedfactor IX gene transfer to skeletal muscle in patientswith severe hemophilia B.  Blood   2003;  101 : 2961–72.MEASUREMENT OF VERY LOW LEVELS OF FVIII:C  257   2006 Blackwell Publishing Ltd  Haemophilia  (2006),  12 , 253–257
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