Number 2 2000 Criteria and search for specific assays for active eJIFCC. The Electronic Journal Of plasminogen activator inhibitor 1 (PAI-1) in plasma the International Federation Of Clinical Chemistry And Laboratory Medicine C. Kluft * Gaubius Laboratory, TNO-PG P.O. Box 2215 2301 CE Leiden The Netherlands Phone: +31. 15181497 Fax: +31.715181904 E-Mail: email@example.com * on behalf of a working group within the Subcommittee on Fibrinolysis, of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis (P.J. Declerck, J. Jespersen, J. Gram, C.Kluft) and the active participants, see acknowledgement (PAI- Criteria and search for specific assays for active plasminogen activator inhibitor 1 (PAI-1) in plasma How to cite this article: Criteria and ods failed to detect sufficiently low activity in depleted plasma and were sensitive to added search for specific assays for active PAI-2. 10 methods were sensitive to interference by endogenous t-PA as revealed by additions of plasminogen activator inhibitor 1 t-PA. Not all methods could adequately measure In this article in acidified plasma. Two methods were fulfilling • Proficiency testing of enzymatic all the criteria (PAI-1) in plasma. www.ifcc.org/ methods for plasminogen activator inhibitor 1 (PAI-1) Conclusions showed a systematic variability ejifcc/vol12no2/PAI-1assays.htm Most methods were not specific for PAI-1 (enz. indicating the aspecificity of procedure) in acidified plasma. Definition of cri- some methods. Abstract teria and test methods by experts of the ISTH/ SSC proved a valuable concept, according to the Background exercise undertaken. Introduction Proficiency testing of enzymatic methods for plasminogen activator inhibitor 1 (PAI-1) Proficiency testing of assays for the fibrinolytic showed a systematic variability indicating the as- variable Plasminogen activator inhibitor 1 (PAI- pecificity of some methods. 1, imm; procedure, enz; procedure) has been executed by collaborative studies within the Methods framework of the SSC (Scientific and Standardi- sation Committee) of the ISTH (International To define and detect specificity of enzymatic Society on Thrombosis and Haemostasis) and methods for PAI-1, experts of the ISTH/SSC showed large interlaboratory and intermethod subcommittee defined criteria and test samples variation (1,2). to check the criteria. 16 samples were prepared In particular the results of studies on the activity to test (a) specificity in depleted plasma, (b) in- methods for PAI-1 showed a systematic variation terference by added PAI-2 or PAI-3, (c) interfer- which could not expected to be solved by har- ence by added tissue-type plasminogen activator monising with a suitable standard. It was de- (t-PA), (d) performance in dose response. To ex- cided that an effort was required to define crite- ercise the test procedure participants were re- ria for specificity of enzymatic PAI-1 methods. cruited via the subcommittee, literature and The situation was more complex since many company data. Coded samples were distributed methods were in use both as commercial meth- to participants. The NIBSC standard for PAI-1 ods and in-house methods and documentation activity was included for the normalisation of re- was insufficient for decisions on specificity. It sults. Adherence to the predetermined criteria was decided that in addition to defining criteria was judged blind in the ISTH/SSC subcommit- an exercise would be executed by providing a set tee meeting. of samples to test the methods for the specificity Results claimed or theoretically expected. The present report, in addition to defining crite- In total 17 laboratories with 15 different assay ria and test samples describes an exercise of ex- methods participated in the study. Methods perimental testing and decisions based on the were based on four detection principles. 8 meth- data and criteria. The data have thus far only Number 2 2000 eJIFCC The Electronic Journal Of The International Federation Of Clinical Chemistry And Laboratory Page 2 Medicine (PAI- Criteria and search for specific assays for active plasminogen activator inhibitor 1 (PAI-1) in plasma been presented without the identification of the Assay 2 is an assay based on the measurement of methods involved. t-PA/PAI-1 complexes present in the sample be- Materials and Methods fore addition and after addition of an excess of t- PA (10). The difference between the two values Samples: reflects the amount of active PAI-1. The study was performed by Dr W. Nieuwenhuizen and R. Sample A was a PAI-1-depleted plasma sample Laterveer using a prefinal version without addi- prepared by immunoadsorption of plasma as tion of PPACK and not yet adapted for use on previously described (3); samples B, C and D acidic plasma as in the reference (10); detection were prepared by adding a defined amount of limit 0.4 ng/ml. purified reactivated recombinant PAI-1 to de- IFCC Technical Secretariat, Assay 3 measures active PAI-1 by addition of ex- pleted plasma (4). 30 Rue Lionnois, cess single chain t-PA and measurement of t-PA- F-54000 Nancy, Samples E and F were prepared by adding to im- PAI-1 complex in both the original and treated France munodepleted plasma a defined amount of re- samples. Active PAI-1 is determined by subtrac- combinant PAI-2 (Delta Biotechnology Ltd, tion and expressed as ng/ml (11). N. Booth and Phone: +33 3 83 35 26 16 Nottingham, UK; batch 25/7; purity >95%) A.M. Croll performed the study. Fax: +33 3 83 32 13 22 (plasma dilution < 1%), or PAI-3 / Protein C Email: Chantal.Thirion@ifccts.u- Assay 4 is a titration assay of PAI-1 in the sample inhibitor (Technoclone GMBH, Vienna, Aus- with a series of two-chain melanoma t-PA con- tria) (plasma dilution 30%). centrations according to Verheijen et al (12) us- ing CNBr fragments to stimulate t-PA activity “Providing leadership in clinical Platelet-poor plasma for sample G (see table I) and measurement of plasmin/chromogenic sub- chemistry and clinical laboratory was prepared as a pooled plasma from blood col- service“. strate activity. Results are expressed in t-PA in- lected from 15 volunteers in 0.129 mol/l of so- ternational units calibrated against NIBSC dium citrate (1:10), kept on ice and centrifuged 83/517. C.Kluft and P. Meijer performed the within 30 minutes at 2000 g for 20 minutes at study. 4_C. PAI-1 was inactivated by incubation of the pooled plasma for 24 hours at 37_C according Assay 5 is Coatest® PAI from Chromogenix AB, Visit the IFCC web to (5). Molndal, Sweden and measures residual single site at www.ifcc. chain t-PA after incubation with plasma, by org Stabilyte plasma for samples H,I,J,K was pre- means of chromogenic substrate activity of plas- pared from blood of volunteers collected in Sta- min formed in 50 minutes in the presence of a bilyte®tubes (6)(Biopool AB Umea, Sweden) fibrin-derived stimulator (13). S. Rosen and L. and handled as above. PAI-activity was initially Wejkum performed the study on lot X0074 em- assessed using method 4, and retrospectively as- ploying the manual version in tubes. Results are signed using method 14 and expressed in U/ml expressed in AU/ml; 1 AU /ml PAI activity be- (NIBSC 87/512). Two samples with 7 U/ml ing defined as the amount required to inhibit 1 and 15 U/ml were used. Samples I and K were IU/ml t-PA. spiked with a specified amount of concentrated two-chain melanoma tissue-type plasminogen Assay 6 is SpectrolyseÔ /pl Biopool AB, Umea, activator (t-PA) (7) (plasma dilution < 1%) with Sweden; a two-stage indirect enzymatic assay as- a value assigned according to t-PA activity assay sessing residual melanoma single chain t-PA after using method 4. incubation with plasma (14-16). t-PA is meas- ured after an acidification step to destroy plasma Samples L and M were NIBSC 87/512 and plasmin inhibitors and by its ability to generate 92/654 from the National Institute for Biologi- plasmin activity on a chromogenic substrate in cal Standards and Control, Potters Bar, UK and the presence of the stimulator poly-lysine. Re- were provided as lyophilised samples. NIBSC sults are expressed in units defined as the 87/512 had a certified value of 25 IU/ml; amount of PAI that inhibits 1 IU of human sct- 92/654 was certified later with 27,5 IU/ml (8). PA calibrated against NIBSC 83/517. A Takada Methods: and T Urano performed the study using v1-1, lot 1102025. Assay 1 is an assay based on the measurement of t-PA/PAI-1 complexes present in the sample be- Assay 7 is also SpectrolyseÔ /pl (see assay 6) and fore addition and after addition of an excess of t- performed by I. Juhan-Vague and J. Ansaldi us- PA (9). The difference between the two values ing v1-1, lot 1102025. reflects the amount of active PAI-1. Assay was Assay 8 is a Bioimmunoassay employing anti- performed by P.J. Declerck and I. Knockaert us- PAI-1 IgG coated on wells, incubation with the ing lot MA-15H12/MA-62E8-HRP, detection sample, incubation after washing with two-chain limit 1 U/ml. t-PA and a subsequent assay of residual t-PA ac- Number 2 2000 eJIFCC The Electronic Journal Of The International Federation Of Clinical Chemistry And Laboratory Page 3 Medicine (PAI- Criteria and search for specific assays for active plasminogen activator inhibitor 1 (PAI-1) in plasma tivity by its ability to convert Glu-plasminogen assessed by the activity of plasmin formed on H- D-Val-Leu-Lys-pNA. One unit is defined as the amount inhibiting 1 unit of two-chain t-PA. K. Okada and O. Matsuo performed the study. Assay 9 is the simplified version of the SOFIA- PAI assay (17) measuring residual melanoma sct- PA after incubation with plasma. Residual t-PA is bound to solid-phase fibrin. t-PA activity is assessed by plasmin activity measured on a chro- mogenic substrate after addition of Glu- plasminogen. PAI activity is expressed in units defined relative to single-chain t-PA calibrated against NIBCS 83/517. The assay is not de- signed for acidified plasma. E. Angles-Cano and S. Loyau performed the study. Assay 10 is TC® Actibind-PAI-1 from Techno- clone GMBH, Vienna, Austria and is based on the immobilisation of functionally active t-PA to plates by means of a monoclonal antibody. PAI- 1 in the test sample binds to t-PA and is then quantified using labelled monoclonal anti-PAI-1 antibody. Results are expressed in U/ml of a PAI Figure 1 standard; detection limit 4.0 U/ml. B.R. Binder and R. Beckmann performed the study with lot Eriksson, P.Falk and A. Hansevi. 5 9 3 o f T C 1 6 0 7 0 . Assay 14 is an u-PA-PAI-1 complex ELISA (20), Assay 11 is TC® R Actibind-PAI-1 CL , which that assesses the u-PA complex formed after in- is a prototype kit not further developed, based cubation with added u-PA. Assay with lot on method 10, however, with t-PA cross-linked 2F5013 was performed by M.Philips and B. to the monoclonal antibody, using a PAI-1 stan- Lillethorup and results were reported in pmol/l dard calibrated according to NIBSC 87/512. active PAI-1; the experience is that when analyz- Assay 12 is TC® PAI activity kit from Techno- ing NIBSC 83/517 29 fmol equals 1 IU. clone GMBH, Vienna, Austria. And is based on Assay 15 is similar to assay 15 with reagents ob- the inhibition of rt-PA added to plasma. Re- tained from Novo Nordisk, Baegsvaerd, Den- maining t-PA activity is determined by its Glu- mark and performed by E. Eriksson, P.Falk and plasminogen activating properties stimulated by A. Hansevi with lot 774-776. CNBr-fragments of fibrinogen and a synthetic plasmin substrate. Plasmin inhibitor is quenched Assay 16 is Stachrom® PAI , from Diagnostica by a special reagent.(18). Stago, Asnières-sur-Seine, France. After incuba- Results are expressed in U/ml of a PAI standard. tion of the plasma with u-PA, the excess u-PA is B.R. Binder and V. Carroll performed the study measured by its capability to generate plasmin in with lot 593 of TC 15070. The method was not an environment with inhibitors for plasmin in- suitable for Stabilyte plasma and results on sam- hibitor and alpha-2-macroglobulin (21).The as- ples H-K should be interpreted accordingly. The say was run on a semi-automated device ST 888, high recording in PAI-depleted samples in this by F. Nicham and N. Barat with lot 922190. study was questioned in view of in house results Assay 17 is Berichrom® R PAI from Behring- and previous results (1). The mixture of single werke AG, Marburg, Germany, an assay which and two-chain rt-PA was later replaced by a sin- employs u-PA as the target enzyme for PAI in gle chain preparation to reduce sensitivity for plasma and measurement of uninhibited u-PA PAI-2 (19). via plasminogen/plasmin conversion and subse- Assay 13 is a two stage method with incubation quent cleavage of a chromogenic plasmin sub- of plasma with an excess of single-chain t-PA fol- strate in an environment with oxidative inactiva- lowed by assay of residual t-PA enzymatic activ- tion of plasmin inhibitor (22,23). The study was ity with a plasminogen/chromogenic plasmin performed by H. Keuper and P. Lenz with a substrate assay utilizing poly-D-lysine as a t-PA two-point method ; lot 25258 (test version). Re- stimulator (16). Values are expressed in IU/ml sults are expressed in u-PA IU/ml which accord- relative to t-PA. The study was performed by E. ing to the experience is 1/5 to 1/6 of the t-PA Number 2 2000 eJIFCC The Electronic Journal Of The International Federation Of Clinical Chemistry And Laboratory Page 4 Medicine (PAI- Criteria and search for specific assays for active plasminogen activator inhibitor 1 (PAI-1) in plasma Table I inhibiting units. Sample code Sample characteristics Results A PAI-1 immunodepleted Criteria for specificity of methods for active PAI- 1 in plasma B A + 4.6 U/ml r-PAI-1 In plasma we can expect different molecular C A + 17.0 U/ml r-PAI-1 forms of PAI-1, such as inactivated PAI-1 D A + 55.8 U/ml r-PAI-1 (originating from platelets, or generated sponta- neously by time-dependent thermal inactivation E A + 50 ng/ml r-PAI-2 of active PAI-1), active PAI-1 and PAI-1 in com- plex with proteases, notably t-PA (plasminogen F A + 5.5 _g/ml PAI-3 activator, tissue-type) or u-PA (urokinase-type G PAI-1 inactivated by incubation at 37_C PA). Besides this complexity we can expect that other inhibitors in plasma compete for the target H Stabilyte plasma (PAI activity 7 U/ml) protease (t-PA or u-PA) used in a test principle. Another complexity was the co-occurrence of ac- I H + 4 IU/ml tc-tPA tive t-PA besides PAI-1 in plasma. This t-PA J Stabilyte plasma (PAI activity 15 U/ml) could compete with the assay of activity or con- tinue to react with PAI-1 after blood sampling. K J + 7 IU/ml tc-t-PA To avoid the latter, a method was introduced L NIBSC standard 87/512 (25 U/ml) around 1988-1990 to acidify blood as soon as possible after its collection or to use acid antico- M NIBSC standard 92/654 (unlabelled) agulants to achieve a pH of around 6 (6,24). This "new" anticoagulation procedure compli- cates the situation as well, since not all assay Specific: Dose-response of added PAI-1 to methods were designed for or adapted to the use plasma (0; 4.6; 17; 55.8 U/ml) should be of high o f a c i d i f i e d p l a s m a . quality (grading A = r > 0.999; grading B = r > In figure 1 the situation in plasma is summarised 0.9; grading C = r < 0.9) and in particular it should be noted that we aim The criteria (1a, 1b, 2, 3) were to be independ- to measure active PAI-1 and not the residual ac- ently applied and any C should result in exclu- tivity of PAI-1. sion and A and B both in inclusion. The crite- There were four test principles involved in the rium on performance was limited to criteria for methods evaluated (see table II) and criteria fo- the dose-response curve and not intended at this cus on these principles. The criteria were estab- stage to evaluate whether the coefficient of varia- lished during the Meeting of the ISTH/SSC tion was half of that of the total, including bio- subcommittee on Fibrinolysis in 1992. logical variation, which should also be evaluated (25). C r i t e r i u m General : No interference by other plasma Samples to evaluate adherence 1 a a n d b : (inhibitory) c o m p o n e n t s to the criteria Specific 1a: Immunodepleted and thermally in- activated plasma should have an activity < 5% of The following samples were prepared and in- that of NIBSC standard 87/512 (grading A = cluded in the set that was distributed: both < 5%; grading B = only one < 5%; grading C = b o t h > 5 % ) r-PAI-1 = recombinant PAI-1; Specific 1b: Added PAI 2 or PAI 3 added to im- r-PAI-2 = recombinant PAI-2; munodepleted plasma should have an activity < tc-t-PA = two chain melanoma 5% of that of NIBSC standard 87/512 (grading t-PA. A = both < 5%; grading C = one or two < 5%) Samples were coded and frozen and distributed C r i t e r i u m 2 : on dry ice except for the NIBSC standards General: No interference by endogenous t PA which were freeze-dried. The original set was lar- Specific: Added t PA to two (acid) plasmas with ger including additions of r-PAI-1 to buffer, but different PAI-1 activity level should not reduce these were later excluded. recorded PAI-1 activity with an amount > 20% of the initial activity (Grading A = mean reduc- Procedure of the evaluation and tion < 20%; grading C = mean reduction > 20%). follow- follow-up. An invitation to participate voluntarily (by direct Criterium 3: Precision / performance Number 2 2000 eJIFCC The Electronic Journal Of The International Federation Of Clinical Chemistry And Laboratory Page 5 Medicine (PAI- Criteria and search for specific assays for active plasminogen activator inhibitor 1 (PAI-1) in plasma mail) to volunteers responding at the subcom- ganisers, the results were not decoded on any oc- mittee meeting, to scientists of original publica- casion and were only subject to consideration by tions of method development, method evalua- the participants for their own method / informa- tion and/or modification (in total 33) and com- tion. The selection of a potential reference pany scientists responsible for commercial meth- method and potential recommendation within ods (in total 14) resulted in 18 responses con- the framework of the SSC was not considered cerning 16 different methods. The general re- appropriate. The present manuscript is the first quirement and criteria were stated in a letter in decoded account of the results. advance. Results of the exercise Thus evaluation was confined to expert laborato- ries or laboratories that had designed a test and a) General aspects were informed about the aims and procedures. It had been decided to perform the analysis From_the 18 applicants, 17 continued co- blind in two ways (a) the test samples were operation and received 16 coded samples. From coded for participants (b) the methods were these sixteen samples, thirteen (see table I) were coded for discussion in the SSC subcommittee used for evaluation. On retrospect, the samples meetings. with PAI-1 added to buffer were excluded from evaluation due to poor and erratic recovery of The results of the evaluation were presented added PAI-1 evident from all data. coded at the SSC subcommittee on fibrinolysis meeting in 1993, New York and on the basis of As two independent participants participated per Table II the criteria and prede- termined judgement, two methods only ful- filled all criteria (see be- low). Subsequently, individ- ual results were distrib- uted to participants for comment, and to ac- knowledge that the sin- gle evaluation of each assay method (except in two cases) might have been subject to inciden- tal error (misfortune) or that the engineered samples might have cre- ated unexpected prob- lems. For instance the use of acidified plasma (pH 6) was quite re- cent. Additional data were welcomed. The reactions have been taken into account. No- tably, the evaluation with added recombi- nant PAI-2 was at vari- ance with the experi- ence of some investiga- tors: this issue was not resolved. After the formal presen- tation of coded results and the evaluation of comments by the or- Number 2 2000 eJIFCC The Electronic Journal Of The International Federation Of Clinical Chemistry And Laboratory Page 6 Medicine (PAI- Criteria and search for specific assays for active plasminogen activator inhibitor 1 (PAI-1) in plasma Table III two methods, the whole procedure could be evaluated. For method 6 and 7 and for 14 and 15 (see table II) the data showed excellent agree- ment as expressed by the mutual correlation of all data r>0.97. b) Method principles The methods evaluated in the present study had different principles for detecting the active PAI- 1 . The principles involved either addition of t-PA to the samples (13 cases) or addition of u-PA (4 cases): The t-PA preparation could be a one- or t w o - c h a i n m o l e c u l e . The detection principle involved the measure- ment of the complex of PAI-1 formed with the added enzyme (7 cases). In view of the normal, endogenous presence of t-PA*PAI-1 complex the increase in the amount of complex was evalu- ated. In the case of u-PA addition it was as- sumed that normally no u-PA*PAI-1 complex was circulating, restricting the use to normal situations where this applies. The detection principle involved in 10 cases the detection of the excess of added enzyme, either t-PA (8 times) or u-PA (2 times) In Table II, the methods are arranged and num- bered according to the principles of assay as mentioned above. As can be observed from the table as well, the units in which the results are provided differ, despite the availability of stan- d) Criterium 1: absence of interference by other dard NIBSC 87/512 (assigned value 25 U/ml). plasma (inhibitory) components For comparison, the results of the participants for the standard in their own units is given in The evaluation on criterium I could not use the the table. It illustrates the variability existing at samples with buffer matrix due to poor recovery/ that time in reporting data on PAI-1 activity. stability of such samples as concluded retrospec- tively, preventing direct evaluation of matrix in- Assays are identified by number and described in terferences materials and methods. AU = arbitrary units. Interference by plasma components and other c) Normalization for evaluation. inhibitors was evaluated by For evaluation all results were normalised rela- 1) the results in immunodepleted plasma and a tive to the included sample of NIBSC 87/512 pooled citrated plasma incubated overnight at and presented in percentages of this standard 37_C to inactivate PAI-1 (activity-depleted) throughout the manuscript, unless specified oth- 2) the results of assay in immunodepleted erwise. For validation of this procedure, for all plasma supplemented with PAI-2 or PAI-3 criteria, normalisation was also exercised with Results are summarised in table III in three cate- the other standard NIBSC 92/654 and with one g o r i e s I - I I I . of the frozen samples ( Stabilyte sample H, see Category I concerns methods with a combina- table I) and similar results and conclusions were tion of a low recording in depleted plasmas obtained in all cases. For nine methods with an (criteria A = both < 5%) and low interference by excellent analytical performance (dose-response PAI-2 (criterium A = below 5%). curve with r> 0.999) the two NIBSC standards Category II does not fulfil criteria A and/or B were compared showing close agreement on an but is less clearly aberrant compared to category activity of NIBSC 92/654 of 86 % of that of I I I NIBSC 87/512 (interquartile range: 81-96, stan- Category III is clearly not adequate due to crite- dard deviation 18%). rium C applying to depleted plasma or the addi- Number 2 2000 eJIFCC The Electronic Journal Of The International Federation Of Clinical Chemistry And Laboratory Page 7 Medicine (PAI- Criteria and search for specific assays for active plasminogen activator inhibitor 1 (PAI-1) in plasma tion of PAI-2. f) Criterium 3: Dose-response curve quality There was no sensitivity to PAI-3; only occa- As shown in Table IV, 7 assays showed an excel- sional values did not fulfil criterium A. lent dose-response (r>0.999) while 4 performed e) Criterium 2: Absence of interference by en- poorly (r< 0.9). The amount of sample provided dogenous free t-PA did not allow for dilution experiments, causing in some cases a poor performance for the higher To test absence of interference by endogenous value of PAI-1 in sample D. active t-PA, the procedure was chosen to add to Stabilyte plasma (where added t-PA is preserved g) Overall scoring and does not react with endogenous PAI-1 in When using either exclusion or inclusion as an the sample) an additional amount of t-PA. This approach only methods 10 and 14 were not ex- extra amount should not interfere with the assay cluded. Grading C on criterium C did not con- of the PAI-1 activity in the sample. The addition tribute independently to any exclusion. to plasma H of 4 IU/ml t-PA and to plasma J of 7 IU/ml t-PA would be comparable to about Discussion 50% potential neutralisation of the endogenous The study presented focused on the specificity of PAI-1 amount. methods for active PAI-1 in acidified plasma. For the evaluated methods for PAI-1 activity we As shown in Table IV, most methods show sig- showed that the majority was not specific ac- nificant reduction in the recording of PAI-1 ac- cording to the tests and criteria applied. Only 2 tivity following the addition of t-PA. According out of 15 methods were potentially specific. to the criteria of change <20% only 4 methods The three major problems causing these results fulfilled the criterium, and out of these only two were (a) the sensitivity of about half of the meth- methods from category I and II from table III. ods to interference by another inhibitory factor Number 2 2000 eJIFCC The Electronic Journal Of The International Federation Of Clinical Chemistry And Laboratory Page 8 Medicine (PAI- Criteria and search for specific assays for active plasminogen activator inhibitor 1 (PAI-1) in plasma in plasma as evident from the results in PAI-1 specificity and to one laboratory, for most meth- depleted plasma, (b) the fact that some methods ods and results should be confirmed in subse- were not adapted to the use of acid or Stabilyte quent more elaborate and detailed experiments plasma , and (c) the sensitivity of many methods for definite conclusions. to endogenous t-PA as revealed by the addition The process in the subcommittee of fibrinolysis of extra t-PA. In addition, most methods aimed of the ISTH/SSC resulted in an increased aware- to assess elevated PAI-1 and performed subopti- ness of the importance of specificity of methods mally at very low levels (cf 1). and the experimental documentation, especially The presence of an interfering plasma factor had for standardisation and when a reference method been described previously (5,26); the identity of is required. In addition it seemed adequately this interfering factor is still unknown. The sen- possible in all methods to use the NIBSC stan- sitivity to this interference coincided with sensi- dards, though, this issue has not been formally tivity to the addition of PAI-2. The effect of investigated. It should be recognised that the PAI-2 was debated in subsequent correspon- evaluation did not focus on possible matrix in- dence with some participants and at variance terference. An inventory on this aspect in the with their experience. This should be noted, but present exercise was not possible because of the was not further evaluated. problems with the analyte added to buffer: the The sensitivity to added t-PA was previously not samples were excluded from evaluation. In fu- considered a major aspect to circumvent since ture evaluations of these aspects require addi- various methods aimed at studying elevated PAI- tional attention. 1 where endogenous t-PA is a minor compo- The ISTH/SSC can be considered a suitable ex- nent. Also the level of endogenous active t-PA pert forum for the definition of criteria and test- was underestimated before the introduction of ing principles in haemostasis. The testing as exe- acidification of blood to inhibit in vitro interac- cuted for the method for PAI-1 activity was seen tion of t-PA and PAI-1. At present it is state-of- as a single exercise and in future the responsibil- the art to use acidified plasma to avoid in vitro ity of parties who develop the methods and stan- interactions and to attempt to measure active dards. PAI-1 (see figure 1). This allows the use of methodology for all purposes independent of the Acknowledgements t-PA status. It can be observed that methods us- Dr. P.J. Declerck (Immunodepleted plasma, ing u-PA as target enzyme for PAI-1 were rela- rPAI-1), Dr. B.R. Binder (PAI-3), Dr D.J. Bal- tively insensitive to endogenous t-PA. lance (rPAI-2) and Dr P.J. Gaffney (NIBSC standards) are gratefully acknowledged for sup- The two methods that fulfilled the criteria pro- ply of materials and samples. Ing. P. Meijer is vided specificity for PAI-1 by the use of specific gratefully acknowledged for sample preparation antibodies either to immobilise t-PA and detect a n d d i s t r i b u t i o n only newly formed PAI-1-t-PA or to detect spe- The following groups are acknowledged for their cifically the formation of the u-PA*PAI-1 com- voluntary participation in the analysis: plex. In method with u-PA the excess of u-PA Dr S. Thorsen, M. Philips and B. Lillethorup, used was apparently sufficient to avoid interfer- Rigshospitalet, Section for Hemostasis & ence by endogenous t-PA. Thrombosis, Dept of Clinical Biochemistry, Co- In later development also method 2 solved the p e n h a g e n , D e n m a r k problem of endogenous t-PA by the addition of Dr. P.J. Declerck and I. Knockaert, Laboratory PPACK (10). Later assay formats with the prin- of pharmaceutical biology and phytopharmacol- ciple of methods 8 and 10 maintained a pH 6 ogy, Institute for Pharmaceutical Sciences, Leu- during binding of the PAI-1, using acidified v e n , B e l g i u m plasma. Dr. W. Nieuwenhuizen and R. Laterveer, Dept The results of the exercise demonstrate the value of Lipids and Endothelium, Gaubius Labora- of the criteria and test samples for the methods tory, IVVO-TNO, Leiden, The Netherlands evaluated. It is possible that new criteria and test Dr. N. Booth and A.M. Croll. Dept of Molecu- samples are necessary when other assay principles lar and Cell biology, University of Aberdeen, or standards are introduced. It should be noted Marischal College, Aberdeen, Scotland that the exercise was not a complete evaluation; Dr C.Kluft and P. Meijer. Gaubius Laboratory, for instance matrix effects and analytical variabil- IVVO-TNO, Leiden, the Netherlands ity relative to the total variability were not as- Dr S. Rosen, E. Ersdal-Badju and L Wejkum. s e s s e d . Chromogenix AB, Molndal, Sweden The exercise was limited to the evaluation of a Dr A. Takada and T. Urano.Dept of physiology, limited number of samples for each aspect of Number 2 2000 eJIFCC The Electronic Journal Of The International Federation Of Clinical Chemistry And Laboratory Page 9 Medicine (PAI- Criteria and search for specific assays for active plasminogen activator inhibitor 1 (PAI-1) in plasma Hamamatsu University School of Medicine, plasminogen activator from human melanoma Hamamatsu, Shizuoka, Japan. cells. In: Mizrahe A, van Wezel AL, eds. Ad- Dr I. Juhan-Vague and J. Ansaldi. Laboratoire vances in biotechnological processes, 2nd ed. d'hematologie, Hôpital d'adultes Timone et New York: AR Liss, 1983:97-110. Universitaire de Marseille, Marseille, France. 8) Gaffney PJ, Edgell TA. The international Dr K. Okada and O. Matsuo. Dept of Physiol- standard for plasminogen activator inhibitor?1 ogy, Kinki University School of Medicine, Osa- (PAI?1) activity. Thromb Haemost 1996;76:80? kasayama City, Japan. 3. Dr. E. Angles-Cano and S. Loyau. INSERM U.143, Institut de pathologie cellulaire, Hôpital 9) Declerck PJ, Verstreken M, Collen D. An im- de Bicetre, Paris, France. munofunctional assay for active plasminogen ac- IFCC Technical Secretariat, Dr. B.R. Binder, R. Beckmann and V. Carroll. tivator inhibitor-1 (PAI-1). Fibrinolysis 1988;2 30 Rue Lionnois, Dept of Medical Physiology, Lab. for Clinical suppl. 2:77-8. F-54000 Nancy, Experimental physiology, University of Vienna, 10) Nieuwenhuizen W, Laterveer R, Hoegee B. France V i e n n a , A u s t r i a . An enzyme immunoassay for the simultaneous Phone: +33 3 83 35 26 16 Dr E. Eriksson, P. Falk and A. Hansevi. Fibri- determination of active type-1 plasminogen acti- Fax: +33 3 83 32 13 22 nolyslab, Dept of Surgery , East Hospital, Goth- vator inhibitor (PAI-1), and t-PA/PAI-1 com- Email: Chantal.Thirion@ifccts.u- e n b u r g , S w e d e n . plexes. Blood Coagul Fibrinolysis 1995;6:520-6. Dr G. Contant, F. Nicham and N. Barat. Serbio Laboratory, Gennevilliers, France. 11) Booth NA, Croll A, Bennett B. The activity Dr H. Keuper and P. Lenz. Dept of Coagulation of plasminogen activator inhibitor-1 (PAI-1) of “Providing leadership in clinical and Fibrinolysis diagnostics, Behringwerke AG, human platelet. Fibrinolysis 1990;4 supp 2:138- chemistry and clinical laboratory 40. service“. Marburg, W-Germany. References 12) Verheijen JH, Chang GT, Kluft C. Evidence for the occurrence of a fast?acting inhibitor for 1) Gram J, Declerck PJ, Sidelmann J, Jespersen tissue?type plasminogen activator in human J, Kluft C. Multicentre evaluation of commercial plasma. Thromb Haemost. 1984;51:392?5. Visit the IFCC web kit methods: plasminogen activator inhibitor ac- site at www.ifcc. tivity. Thromb Haemostas 1993;70:852?7. 13) Wejkum L, Chmielewska J. A new adapta- tion of Coatest PAI for measurement of low in- org 2) Declerck PJ, Moreau H, Jespersen J, Gram J, hibitor concentrations in plasma. Fibrinolysis Kluft C. Multicenter evaluation of commercially 1990;4:130. available methods for the immunological deter- mination of plasminogen activator inhibitor?1 14) Chmielewska J, Ranby M, Wiman B. Evi- (PAI?1). Thromb Haemostas 1993;70:858?63. dence for a rapid inhibitor to tissue plasminogen activator in plasma. Thromb Res 1983;31:427- 3) Declerck PJ, De Mol M, Alessi M-C, Baud- 36. ner S, Paques GP, Preissner KT, Muller- Berghaus G. Collen D. Purification and charac- 15) Chmielewska J, Wiman B. Determination of terization of a plasminogen activator inhibitor 1 tissue plasminogen activator and its fast inhibitor binding protein from human plasma. J Biol in plasma. Clin Chem 1986;32:482-5. Chem 1988;263:15454-61. 16) Eriksson E, Ranby M, Gyzander E, Risberg 4) Alessi M-C, Declerck PJ, De Mol M, Nelles B. Determination of plasminogen activator in- L, Collen D. Purification and characterization of hibitor in plasma using t-PA and a chromogenic natural and recombinant plasminogen activator single-point poly-D-lysine stimulated assay. inhibitor 1(PAI-1). Eur J Biochem Thromb Res 1998;50:910101. 1988;175:531-40. 17) Angles-Cano E, Masson-Lunven C, 5) Kluft C, Jie AF, Sprengers ED, Verheijen JH. Gaussem P. Development of an internal stan- Identification of a reversible inhibitor of plasmi- dard for plasminogen activator inhibitor-1 PAI-1 nogen activators in blood plasma. FEBS Lett. and its use in a simplified assay for measuring 1985;190:315?8. PAI-1 activity in human plasma. Fibrinolysis 1990;4 supp 2: 127-9. 6) Ranby M, Sundell IB, Nilsson TK. Blood col- lection in strong acidic citrate anticoagulant used 18) Korninger C, Wagner O, Binder BR. Tissue in a study of dietary influence on basal tPA ac- plasminogen activator inhibitor in human tivity. Thromb Haemost 1989;62:917?22. plasma: development of a functional assay sys- tem and demonstration of a correlating 7) Kluft C, van Wezel AL, van der Velden Mr=50.000 antiactivator. J Lab Clin Med CAM, Emeis JJ, Verheijen JH, Wijngaards G. 1 9 8 5 ; 1 0 5 : 7 1 8 - 2 4 . Large scale production of extrinsic (tissue-type) Number 2 2000 eJIFCC The Electronic Journal Of The International Federation Of Clinical Chemistry And Laboratory Page 10 Medicine (PAI- Criteria and search for specific assays for active plasminogen activator inhibitor 1 (PAI-1) in plasma 19) Thorsen S, Philips M, Selmer J, Lecander I, Astedt B. Kinetics of inhibition of tissue-type and urokinase-type plasminogen activator by plasminogen-activator inhibitor type 1 and type 2. Eur J Biochem 1988;175:33-9. 20) Philips M, Juul A-G, Selmer J, Lind B, Thorsen S. A specific immunological assay for functional plasminogen activator inhibitor 1 in plasma. Standardized measurements of the in- hibitor and related parameters in patients with IFCC Technical Secretariat, venous thromboembolic disease. Thromb 30 Rue Lionnois, Haemost 1992;68:486-94. F-54000 Nancy, 21) Contant G, Nicham F, Martinoli JL. Deter- France mination of plasminogen activator inhibitor Phone: +33 3 83 35 26 16 (PAI) by a new venom based assay. Fibrinolysis Fax: +33 3 83 32 13 22 1992; 6 supp 3:85-6. Email: Chantal.Thirion@ifccts.u- 22) Stief TW, Lenz P, Becker U, Heimburger N. Determination of plasminogen activator in- hibitor (PAI) capacity of human plasma in pres- “Providing leadership in clinical ence of oxidants: a novel principle. Thromb Res chemistry and clinical laboratory 1988;50:559-73. service“. 23) Stief TW, Lenz P, Becker U, Heimburger N. Functional determination of plasminogen ac- tivator inhibitor (PAI) based on oxidative inacti- vation of alpha-2-antiplasmin: no influence of Visit the IFCC web sample heparin and fibrinogen split products site at www.ifcc. (FSP). Fibrinolysis 1988;2 supp 2:73-4. org 24) Kluft C, Verheijen JH. Fibrinolysis Working Party: Blood collection and handling procedures for assessment of tissue-type plasminogen activa- tor (t-PA) and plasminogen activator inhibitor 1 (PAI-1). Fibrinolysis 1990;4 supp 2:155-61. 25) Fraser CG, Harris EK. Generation and ap- plication of data on biological variation in clini- cal chemistry. Crit Rev Clin Lab Sci 1989;27:409-37. 26) Sprengers ED, Kluft C. Plasminogen activa- tor inhibitors. Blood 1987;69:381?7.