1、 BRITISH STANDARD BS 3985:2003 Haemiglobincyanide (cyanmethaemoglobin) preparation as a standard for spectrometric haemoglobinometry ICS 11.100 BS 3985:2003 This British Standard, was published under the authority of the Standards Policy and Strategy Committee on 10 October 2003 BSI 10 October 2003
2、First published January 1966 Second edition September 1978 Third edition October 2003 The following BSI references relate to the work on this British Standard Committee reference CH/212 Draft for comment 03/107403 ISBN 0 580 42710 2 Committees responsible for this British Standard The preparation of
3、 this British Standard was entrusted to Technical Committee CH/212, In vitro diagnostics, upon which the following bodies were represented: Association of Anaesthetists of Great Britain and Northern Ireland Association of British Healthcare Industries (ABHI) Association of Clinical Biochemists Assoc
4、iation of the British Pharmaceutical Industry Association of the Laboratory Supply Industry (BLWA Ltd.) British Blood Transfusion Society British Diabetic Association British Glass Manufacturers Confederation British In Vitro Diagnostics Association British Society for Antimicrobial Chemotherapy Bri
5、tish Society for Haematology Clinical Pathology Accreditation (UK) Department of Health, Medical Devices Agency Department of Health, Medicines Control Agency Guild of Hospital Pharmacists Institute of Biomedical Science Institute of Quality Assurance Medical Sterile Products Association National In
6、stitute for Biological Standards and Control Parenteral Society Radcliffe Hospital, Oxford Royal College of Pathologists Royal Society of Health Scottish National Blood Transfusion Service United Kingdom National External Quality Assessment Service (UK NEQAS) Coopted members Amendments issued since
7、publication Amd. No. Date CommentsBS 3985:2003 BSI 10 October 2003 i Contents Page Committees responsible Inside front cover Foreword ii 1S c o p e 1 2T e r m s a n d d e f i n i t i o n s 1 3M a t e r i a l 1 4 Concentration 1 5P u r i t y 2 6E x p i r y d a t e 2 7 Inscriptions on ampoules 2 8 Ins
8、criptions on containers 3 Annex A (informative) Method for use of standard for estimation of haemoglobin in a blood sample 4 Bibliography 7 Figure 1 Absorption curve for haemiglobincyanide solution 3BS 3985:2003 ii BSI 10 October 2003 Foreword This British Standard has been prepared by Technical Com
9、mittee CH/212. It supersedes BS 3985:1978, which is withdrawn. This British Standard was first published in 1966 and revised in 1978. The present new edition brings it up to date for use in the determination of haemoglobin by spectrometers which require calibration if the results are to be obtained
10、in absolute terms. A reliable preparation can also be used to check performance of any spectrometer over the specified range of wavelengths and for checking the accuracy of dedicated haemoglobinometers which are pre-calibrated at the factory or rely on artificial standards. This new edition of the s
11、tandard incorporates technical changes only. It does not represent a full review or revision of the standard, which will be undertaken in due course. Converting the haemoglobin from blood into haemiglobincyanide (HiCN), (formerly termed cyanmethaemoglobin) is now the preferred method for preparing a
12、 reference standard as this is stable for at least six years (van Assendelft et al. 1976 1 and 1996 2). The International Council (formerly Committee) for Standardization in Haematology (ICSH) established specifications for this, based on a molar area (decadic) absorbance of 11.0 at a wavelength of
13、540 nm (the absorbance of a solution containing 55.85 mg/l of haemoglobin iron and having a light path length of 1.000 cm) and a molar mass of 64 458/4 = 16 114.5 for human blood and 64 532/4 = 16 133 for bovine blood. These specifications conform to the International Council for Standardization in
14、Haematology standard which was established at the tenth Congress of the International Society of Haematology (ICSH, 1978 3) and later reviewed by an Expert Panel (ICSH, 1995 4). This preparation was adopted by the World Health Organization as an international reference standard and also by the Europ
15、ean Commission Bureau of Reference (Lewis, 1996 5). This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pag
16、es This document comprises a front cover, an inside front cover, pages i and ii, pages 1 to 7 and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued.BS 3985:2003 BSI 10 October 2003 1 1 Scope This British Standard specifies a solution of hae
17、miglobincyanide (cyanmethaemoglobin) for use in blood analysis. NOTE The method of use of the standard specified in this British Standard is given in Annex A. 2 Terms and definitions For the purposes of this British Standard, the following terms and definitions apply. 2.1 transmission factor the rat
18、io of the transmitted radiant flux to the incident radiant flux at a single wavelength 2.2 transmittance the ratio of the transmission factor of the liquid solution to that of the solvent in a cell of the same form and thickness 2.3 absorbance the logarithm to the base 10 of the reciprocal of the tr
19、ansmittance NOTE This definition specifies a quantity which is proportional to the dissolved solute and is not precisely the same as is specified by certain alternative definitions of this term. 2.4 path length the distance traversed by the radiation between two boundary surfaces of the liquid under
20、 test 3 Material The material shall be an aqueous solution of haemiglobincyanide. This shall be prepared from washed and haemolyzed red cells from human or bovine blood, the haemoglobin of which is then converted to haemiglobincyanide. It shall be supplied in a nominal volume of 10 ml in a sealed am
21、poule of amber glass. It shall be sterile and shall not contain any suspended material visible to the naked eye. 4 Concentration The concentration of haemiglobincyanide in the solution shall be calculated from the observed absorbance when a parallel beam of monochromatic light of wavelength 540 nm t
22、raverses a 1.000 0.005 cm path length of the solution. The absorbance of the haemiglobincyanide solution shall be measured on a spectrophotometer, the wavelength scale of which has been calibrated with the aid of the Hg (or H) emission spectrum and on which absorption checks have been performed usin
23、g calibrated glass filters or other means which have been tested by independent standardizing laboratories. The slit width shall be so chosen that the half intensity band-width is less than 1 nm. The cells in which the solution is measured shall be plane-parallel with an inner wall-to-wall distance
24、of 1.000 0.005 cm. The measurements shall be carried out over the temperature range 20 C to 25 C.BS 3985:2003 2 BSI 10 October 2003 The haemoglobin concentration shall be expressed in milligrams per litre and given by one of the following relations. where A standard solution shall contain 700 150 mg
25、/l of haemiglobincyanide. 5 Purity The purity of the solution shall be controlled as follows. a) The shape of the absorption curve between the wavelengths 450 nm and 750 nm shall conform to the absorption curve shown in Figure 1. b) The ratio of the absorbance at 540 nm to that at 504 nm shall be 1.
26、61 0.02. c) The absence of turbidity in the solution shall be confirmed by measuring the absorbance at 750 nm. The absorbance (path length 1 cm) shall be not greater than 0.002. NOTE This measurement should be made relative to deionized or distilled water conforming to the requirements of BS EN ISO
27、3696. 6 Expiry date The life of the solution in its sealed ampoule shall be considered as not greater than 5 years from the date on which the solution is prepared. 7 Inscriptions on ampoules Every ampoule of standard solution shall be labelled with the following information: a) the concentration of
28、haemiglobincyanide in the solution as calculated from Clause 4, expressed to the nearest milligram per litre; b) the makers or vendors name or mark; c) the date of expiry in accordance with Clause 6; d) the number and date of this British Standard i.e. BS 3985:2003 1) ; e) the makers batch number. o
29、r (see below) C is the concentration of haemoglobin in milligrams per litre; is the absorbance of the solution of haemiglobincyanide (HiCN) at a wavelength, , of 540 nm; 16 114.5 is the molar mass of haemoglobin from human blood; and 16 133 is the molar mass of haemoglobin from bovine blood; 11.0 is
30、 the molar area (decadic) absorbance (i.e. the millimolar absorption coefficient of HiCN at a wavelength of 540 nm); 1.000 is the light path in centimetres to an accuracy of three decimal places. 1) Marking BS 3985:2003 on or in relation to a product represents a manufacturers declaration of conform
31、ity, i.e. a claim by or on behalf of the manufacturer that the product meets the requirements of the standard. The accuracy of the claim is solely the claimants responsibility. Such a declaration is not to be confused with third-party certification of conformity. C A HiCN 540 16 114.5 11.0 1.000 - -
32、 = C A HiCN 540 16 133 11.0 1.000 - - = A HiCN 540BS 3985:2003 BSI 10 October 2003 3 8 Inscriptions on containers Each container in which the ampoules are packed shall be labelled as follows: a) with the items a) to e) listed in Clause 7; b) with brief recommendations on storage, i.e. that it should
33、 be kept in the dark and within the temperature range 2 C to 10 C; c) with a recommendation that the solution in an ampoule should be used immediately after the ampoule is opened and should then be discarded. Figure 1 Absorption curve for haemiglobincyanide solution Absorbance Wavelength, nm 0.450 0
34、 -0.050 450 510 504 540 570 630 690 750BS 3985:2003 4 BSI 10 October 2003 Annex A (informative) Method for use of standard for estimation of haemoglobin in a blood sample A.1 General The solution specified in this standard is intended for use in estimation of haemoglobin either directly as haemiglob
35、incyanide (HiCN) by the direct method or indirectly as another haemoglobin derivative. For the direct method blood is diluted in a cyanide-ferricyanide reagent to convert the blood to haemiglobincyanide. In routine practice it may be more convenient to use a non-cyanide diluent, e.g. ammoniated wate
36、r which converts the haemoglobin to oxyhaemoglobin. It is then necessary to prepare a secondary standard of diluted blood, the haemoglobin concentration of which has first to be assigned by the direct method using the standard solution. A.2 Reagent for direct method A suitable reagent has the follow
37、ing constituents dissolved in 1 litre of water 2) : a) potassium ferricyanide, 200 mg; b) potassium cyanide, 50 mg (POISON, USE WITH CARE); c) potassium dihydrogen phosphate, 140 mg; d) a suitable quantity of a colourless surface-active agent (see Note). NOTE 1 ml of Nonidet P40 (VWR International:
38、BDH, Merck, Eurolab) or Triton X-100 (Aldrich) have been found to be satisfactory. If any other similar agent is used, the required conversion time should first be checked. The pH should be 7.0 to 7.4 (checked by pH meter). If stored at room temperature in a brown borosilicate glass bottle, the reag
39、ent keeps for several months, but should be checked regularly. It should not be allowed to freeze as this will inactivate it. WARNING: It is essential that the bottle is labelled to indicate that it contains a hazardous material. A.3 Procedure for direct method A.3.1 Convert the blood sample into a
40、haemiglobincyanide solution by making an accurate dilution of it with the recommended reagent (see A.2) and allowing the diluted sample to stand for 3 min to 4 min. It is usually appropriate to choose the quantities of blood and reagent so that the final concentration of the pigment is 1/201 or 1/25
41、1 of that in the original blood; this ensures that the absorbance is in an optimal part of the scale of the measuring instrument. A.3.2 Using matched 1 cm standard cells or matched tubes and a suitable spectrophotometer or filter photometer (visual or photoelectric), observe the absorbance of the di
42、luted blood solution and the standard, using monochromatic light of wavelength 540 nm or a filter 3)having a sharp absorption minimum at this wavelength. A cell or tube filled with water 4)or the specified reagent should be used to set the instrument, if necessary. 2) Water conforming to the require
43、ments of BS EN ISO 3696 is suitable. 3) An Ilford 625 filter is suitable. 4) Water conforming to the requirements of BS EN ISO 3696 is suitable.BS 3985:2003 BSI 10 October 2003 5 A.4 Calculations for direct method Calculate the concentration of haemoglobin in the blood sample from the following form
44、ula. where Alternatively, a calibration curve may be constructed by plotting the observed absorbance of the standard solution (reference preparation) against its stated concentration and drawing a straight line through this point and the origin. This procedure assumes a strict adherence to the Lambe
45、rt-Beer law and it is advisable to confirm this. When it is necessary to prepare solutions of a concentration different from that provided by the reference preparation, such solutions may be made by diluting the reference preparation with water or the specified reagent. Such dilutions may be used fo
46、r confirming adherence to the Lambert-Beer law. A.5 Indirect method To prepare a secondary standard determine the haemoglobin concentration of an anticoagulated sample of normal whole blood by the haemiglobincyanide direct method as described in A.2, A.3 and A.4. Then dilute the blood 1/201 in ammon
47、iated water (0.4 ml/1 ammonia, specific gravity 0.88, in deionised or distilled water) and assign a haemoglobin value to this secondary standard. This solution is unstable and a fresh one should be prepared every 1 to 2 days. For measuring the haemoglobin of any blood specimen make a dilution (e.g.
48、1/201 or 1/251) in ammoniated water. Read the absorbance of the secondary standard and of the diluted blood in a spectrometer at the same wavelength (540 nm is recommended for oxyhaemoglobin). Calculate the haemoglobin concentration from the following formula: where NOTE Haemoglobin content of blood
49、 may be expressed in terms of substance concentration or in terms of mass concentration. If substance concentration is used it should be expressed in micromols per litre and the elementary entity (monomer or tetramer) needs to be specified by using the notation (Fe) or 4(Fe). If mass concentration is used it should be expressed in grams per litre (in some countries grams per decilitre i
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