1、September 2015 English price group 6No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 11.100.30!%IJf“2383967www.din.d
2、eDIN 58912-1Haemostaseology Determination of the antithrombin activity Part 1: Reference measurement procedure using a synthetic peptide substrate,English translation of DIN 58912-1:2015-09Hmostaseologie Bestimmung der Antithrombin-Aktivitt Teil 1: Referenzmessverfahren mit einem synthetischen Pepti
3、dsubstrat,Englische bersetzung von DIN 58912-1:2015-09Hmostasiologie Dosage de lactivit de lantithrombine Partie 1: Mode opratoire de mesure de rfrence laide dun substrat peptidique synthtique,Traduction anglaise de DIN 58912-1:2015-09SupersedesDIN 58912-1:2000-03www.beuth.deDocument comprises 8 pag
4、esDTranslation by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authoritative.12.15DIN 58912-1:2015-04 2 A comma is used as the decimal marker. Contents Page Foreword 3 1 Scope 4 2 Normative references 4 3 Terms and definitions .4 4 Designation 5 5 Collection
5、and processing of blood.5 6 Procedure .6 6.1 Preparation of samples and reagents6 6.2 Methods 6 6.2.1 Method for thrombin (factor IIa) 6 6.2.2 Method for factor Xa 6 6.3 Reaction mixture 7 6.3.1 Manual method for thrombin (factor IIa) .7 6.3.2 Manual method for factor Xa 11 .7 6.4 Quality control 7
6、7 Report of results 7 Bibliography 8 DIN 58912-1:2015-04 3 Foreword This document has been prepared by Working Committee NA 063-03-05 AA Hmostaseologie of the DIN-Normenausschuss Medizin (DIN Standards Committee Medicine) in cooperation with the Gesellschaft fr Thrombose- und Hmostaseforschung e. V.
7、 (Society for Thrombosis and Haemostasis Research). Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. DIN and/or DKE shall not be held responsible for identifying any or all such patent rights. Amendments The following amendments ha
8、ve been made to DIN 58912-1:2000-03: a) the term antithrombin III has been updated to antithrombin; b) the factor Xa method has been added to the document and the standard, including the terms and definitions, has been revised accordingly; c) the English version of this standard is published separat
9、ely; d) the standard has been editorially revised. Previous editions DIN 58912-1: 1994-06, 2000-03 DIN 58912-1:2015-04 4 1 Scope This standard specifies a procedure for the functional determination of the antithrombin activity (formerly known as antithrombin III AT III) in the form of heparin cofact
10、or activity with regard to thrombin. The purpose of this standard is to establish comparable results for the antithrombin content of a sample. This determination is mainly used in the diagnosis of thrombophilic diathesis, for monitoring of a substitution therapy, and for determining the content in a
11、ntithrombin concentrates. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced doc
12、ument (including any amendments) applies. DIN 58905-1, Haemostaseology Blood collection Part 1: Preparation of plasma from citrated venous blood for coagulation testing DIN 58911-2, Haemostaseology Calibration of measurement procedures Part 2: Photometric measure-ment procedures DIN 58939-1, Haemost
13、aseology Reference plasma Part 1: Requirements, preparation DIN 58963-3, Optical cells for photometric measurements Disposable rectangular spectrophotometric cells Dimensions, requirements 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 antit
14、hrombin activity heparin cofactor activity of antithrombin Note 1 to entry: The influence of other physiological antithrombins (alpha-2-macroglobulin, heparin cofactor II) is minimized by appropriate reaction conditions. 3.2 antithrombin calibration plasma fresh, deep-frozen or lyophilized pooled pl
15、asma obtained from at least ten healthy donors, which is calibrated against an antithrombin reference plasma Note 1 to entry: The antithrombin calibration plasma is used for establishing the reference curve as specified in DIN 58911-2. Note 2 to entry: Dilutions for multi-point calibration are permi
16、tted as long as they match the calibration ratio of an adequately diluted antithrombin reference plasma. 3.3 antithrombin normal activity antithrombin coagulant activity in a fresh normal pooled plasma as specified in DIN 58911-2 Note 1 to entry: The antithrombin normal activity is defined as 100 %
17、or 1 International Unit per millilitre of plasma 4, 5. DIN 58912-1:2015-04 5 3.4 antithrombin reference concentrate lyophilized antithrombin concentrate, the antithrombin activity of which is calibrated against the WHO International Concentrate Standard for antithrombin which is valid at the time of
18、 use Note 1 to entry: The antithrombin reference concentrate is used for calibration purposes and can also be used to establish a reference curve. 3.5 antithrombin reference plasma pooled normal plasma which is stored deep-frozen at 70 C or lyophilized, and which is calibrated against the WHO Intern
19、ational Standard for antithrombin which is valid at the time of use Note 1 to entry: Pooled normal plasma according to, for instance, DIN 58939-1, can be used to make antithrombin reference plasma. Note 2 to entry: The antithrombin reference plasma is used for calibration purposes and can also be us
20、ed to establish a reference curve. 3.6 chromogenic peptide substrate synthetic oligopeptide with a chromophoric group at the C-terminal end, which is cleft by factor Xa or thrombin (factor IIa) 3.7 factor Xa (EC 3.4.21.6)1)proteolytic enzyme which is formed by partial proteolysis of the proenzyme fa
21、ctor X 2 3.8 heparin sodium or calcium salt of a sulfated glucosaminoglycan, that is found in the tissue of humans and animals Note 1 to entry: The characteristic property of heparin is that it prolongs the clotting time of fresh blood 3. Note 2 to entry: Heparin activity is given in International U
22、nits per millilitre (lU/ml) and is defined by the WHO International Standard valid at the time of use. 3.9 thrombin (EC 3.4.21.5)1)proteolytic enzyme which is formed by partial proteolysis of the proenzyme prothrombin Note 1 to entry: The activity of thrombin is generally given in International Unit
23、s per millilitre (IU/ml) 1. 4 Designation Designation of the determination of antithrombin using a chromogenic peptide substrate according to this standard: Determination DIN 58912-1:2015 AT chromogenic 5 Collection and processing of blood The collection and processing of blood shall be carried out
24、as described in DIN 58905-1. 1)Designation according to IUPAC (International Union of Pure and Applied Chemistry). DIN 58912-1:2015-04 6 The plasma sample may be stored up to 8 h after blood collection at a temperature in the range from 18 C to 22 C. If the determination cannot be performed within t
25、his period of time, the plasma sample may be frozen as described in DIN 58905-1. 6 Procedure 6.1 Preparation of samples and reagents The plasma sample shall be pre-diluted in physiological saline solution, whereby the dilution ratio should be in the range of 1:20 to 1:100. The pre-diluted sample sha
26、ll not be stored longer than 1 h at room temperature prior to the determination. Antithrombin concentrates shall be pre-diluted to about 1 IU/ml in a diluent containing 0,1 % human albumin. NOTE Reaction conditions can be modified as long as comparable results are obtained. 6.2 Methods 6.2.1 Method
27、for thrombin (factor IIa) Bovine thrombin shall be used as thrombin, because of its lower affinity for heparin cofactor II. The concentration shall be selected so that about 60 % of the activity of the bovine thrombin is bound by the antithrombin content of a sample with about 1 IU/ml. In the inhibi
28、tion phase, the heparin concentration is at least 1,0 IU/ml, so that thrombin inhibition caused by antithrombin will be completed within the specified reaction time. A TRIS HCl buffer with a pH value of 7,5 to 9,0 shall be used as the reaction buffer. Albumin should be added as a stabilizer for thro
29、mbin, so that a concentration in the buffer in the range of 0,1 % to 1 % is obtained in order to prevent adsorption. Suitable protease inhibitors (e.g. aprotinine) shall be added in order to prevent unspecific reactions. A synthetic chromogenic substrate with a chromophoric group at the C-terminal e
30、nd is used for detecting the reaction. The increase of spectral internal absorbance per minute in the blank should be at least 0,2 under the reaction conditions chosen and at a wavelength of 405 nm. The concentration should be at least 2Km2)or the increase of spectral internal absorbance over time s
31、hould have a linear progress for at least 90 s. The determination is carried out at (37,0 0,5) C. The calibration is carried out as described in DIN 58911-2. Alternatively, the calibration may be carried out using a reference plasma and an enzyme blank of the thrombin-heparin reagent. In this case,
32、an enzyme blank per series shall be established. 6.2.2 Method for factor Xa The factor Xa reagent used shall be of bovine origin. The concentration shall be selected so that about 60 % of the activity of the bovine factor Xa reagent is bound by the antithrombin content of a sample with about 1 IU/ml
33、. In the inhibition phase, the heparin concentration is at least 2,0 IU/ml, so that factor Xa inhibition caused by antithrombin will be completed within the specified reaction time. A TRIS HCl buffer with a pH value of 7,5 to 9,0 shall be used as the reaction buffer. Suitable protease inhibitors (e.
34、g. aprotinine) shall be added in order to prevent unspecific reactions. 2)Km: Michaelis-Menten constant 6, 7, 8, 9, 10 DIN 58912-1:2015-04 7 A synthetic chromogenic substrate with a paranitroaniline group at the C-terminal end is used for detecting the reaction. The increase of spectral internal abs
35、orbance per minute in the blank should be at least 0,2 under the reaction conditions chosen and at a wavelength of 405 nm. The concentration should be at least 2Kmor the increase of spectral internal absorbance over time should have a linear progress for at least 90 s. The determination is carried o
36、ut at (37,0 0,5) C. The calibration is carried out as described in DIN 58911-2. Alternatively, the calibration may be carried out using a reference plasma and an enzyme blank of the factor Xa-heparin reagent. In this case, an enzyme blank per series shall be established. 6.3 Reaction mixture 6.3.1 M
37、anual method for thrombin (factor IIa) The measurement shall be conducted at the wavelength specified in the substrate manufacturers instructions. Disposable cuvettes according to DIN 58963-3 shall be used. The reagent solutions shall be pre-warmed to (37,0 0,5) C. 200 l of diluted plasma shall be p
38、ipetted into a dry cuvette pre-warmed to (37,0 0,5) C. Immediately afterwards, 1,0 ml of thrombin-heparin reagent shall be added and thoroughly mixed with the plasma. After 120 s (completion of inhibition phase), 1,0 ml of chromogenic substrate solution shall be added and mixed. After 10 s, the spec
39、tral internal absorbance is measured. Subsequently, the linear change of spectral internal absorbance is measured by at least three other measurement points at equal time intervals over a period of time not longer than 90 s. 6.3.2 Manual method for factor Xa 11 The measurement shall be conducted at
40、the wavelength specified in the substrate manufacturers instructions. Disposable cuvettes according to DIN 58963-3 shall be used. The reagent solutions shall be pre-warmed to (37,0 0,5) C. 200 l of diluted plasma shall be pipetted into a dry cuvette pre-warmed to (37,0 0,5) C. Immediately afterwards
41、, 200 l of factor Xa reagent shall be added and thoroughly mixed with the plasma. After 90 s (completion of inhibition phase), 200 l of chromogenic substrate solution shall be added and mixed. After 30 s, spectral internal absorbance is measured. Subsequently, the linear change over time of spectral
42、 internal absorbance shall be measured at at least three other measurement points at equal time intervals over a period of time not longer than 90 s. The change of spectral internal absorbance recorded over time is a measure of the antithrombin activity of the sample. Calibration is carried out by m
43、eans of the antithrombin reference plasma or antithrombin reference concentrate. The aforementioned volumes of solutions may be changed proportionally to obtain macro- or micro-assays without altering the measuring conditions or calculation mode. 6.4 Quality control ln addition to the reference curv
44、e, the accuracy of each measuring series shall be ensured by including control plasmas with assigned values within the reference interval and the pathological range. 7 Report of results Antithrombin activity calculated from the determined change of spectral internal absorbance per time unit is given
45、 as a percentage or in International Units per millilitre (IU/ml). DIN 58912-1:2015-04 8 Bibliography 1 WHO International Standard, specialized information of NIBSC (National Institute for Biological Standards and Controls) relevant to the International Standard for human thrombin valid at the time
46、of use 2 Non WHO Reference Material, specialized information of NIBSC (National Institute for Biological Standards and Controls) relevant to the International Standard for factor Xa valid at the time of use 3 Deutsches Arzneibuch (German Pharmacopoeia DAB 10) (obtainable from Deutscher Apotheker-Ver
47、lag, Stuttgart, or Govi-Verlag GmbH, Frankfurt/Main) 4 Kirkwood, T. B. L., Barrowcliffe, T. W., Thomas, D. D. 1980. An international collaborative study establishing a reference preparation for antithrombin III; Thromb. Haemostas. 43, pp. 10-15 5 WHO Expert Committee on Biological Standardization; T
48、hirtieth Report; Technical Report Series 638, WHO, Geneva, 1979, p. 21 6 A.L. Lehninger, Biochemie, Verlag Chemie (1987), pp. 147-174 7 T.W. Goodwin, Structure and activity of enzymes, Academic Press (1964) 8 A. Garen, Biochim. Biophys. Acta, 38 (1960), pp. 470-483 9 Th.E. Barman, Enzyme Handbook, S
49、pringer-Verlag (1969) 10 D. Voet, J.G. Voet, Biochemie, Verlag Chemie (1992), pp. 309-347 11 Khor, B., Van Cott, E. M., 2010. Laboratory tests for antithrombin deficiency. American Journal of Hematology, pp. 947-950 12 WHO International Concentrate Standard for antithrombin which is valid at the time of use
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