DIN SPEC 58961-2013 Technical Report - Thrombocyte diagnostics《技术报告 血小板诊断》.pdf

1、April 2013 Translation by DIN-Sprachendienst.English price group 13No 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 DIN Specifications.ICS 11.100.30Th

2、ere are various procedures for developing a DIN SPEC: This document has been developed in accordance with the Technical Report procedure.!%*.E“2071134www.din.deDDIN SPEC 58961Technical Report Thrombocyte diagnosticsEnglish translation of DIN SPEC 58961:2013-04Fachbericht ThrombozytendiagnostikEnglis

3、che bersetzung von DIN SPEC 58961:2013-04Rapport Technique Diagnostic de thrombocyteTraduction anglaise de DIN SPEC 58961:2013-04www.beuth.deDocument comprises 28 pagesIn case of doubt, the German-language original shall be considered authoritative.01.14 DIN SPEC 58961:2013-04 2 A comma is used as t

4、he decimal marker. Contents Page Foreword 3 1 Scope 4 2 Symbols and abbreviations 4 3 Pre-analytical procedures in platelet function testing .5 3.1 Patient preparation in platelet function testing 5 3.2 Blood extraction.5 3.2.1 Light transmission aggregometry (Borns method) (LTA) 5 3.2.2 Multiple el

5、ectrode aggregometry (MEA) and impedance aggregometry .6 3.2.3 Platelet function analyser (PFA systems) .6 3.3 Determination of platelet count and packed cell volume 6 3.4 Transporting samples .6 3.5 Sample preparation .6 3.5.1 Light transmission aggregometry (Borns method) (LTA) 6 3.5.2 Impedance a

6、ggregometry .7 3.5.3 PFA systems 7 3.6 Reference range .7 4 Activation paths for platelet function testing .7 5 Platelet aggregation methods 9 5.1 Light transmission aggregometry (Borns method) (LTA) 9 5.1.1 General 9 5.1.2 Principle 9 5.1.3 Equipment, materials and reagents .9 5.1.4 Sample preparat

7、ion .9 5.1.5 Procedure .9 5.1.6 Interpretation . 10 5.1.7 Quality control . 16 5.2 Aggregation methods Electrical impedance aggregometry (Cardinals method) and multiple electrode aggregometry (MEA) 16 5.2.1 General . 16 5.2.2 Principle . 16 5.2.3 Equipment, materials and reagents 16 5.2.4 Sample pre

8、paration 17 5.2.5 Procedure 17 5.2.6 Interpretation . 18 5.2.7 Quality control . 19 6 The PFA system 20 6.1 General . 20 6.2 Procedure 20 6.3 Interpretation of results . 21 6.3.1 Expected results . 21 6.3.2 Reference ranges and cut-off values 22 6.4 Clinical applications of PFA test cartridges 23 6.

9、5 Quality control . 23 7 Interfering factors . 23 7.1 General . 23 7.2 Method-specific interfering factors. 24 7.2.1 LTA . 24 7.2.2 Impedance aggregometry 24 7.2.3 PFA systems . 24 Bibliography . 25 DIN SPEC 58961:2013-04 3 Foreword This document has been prepared by Working Committee NA 063-03-05 A

10、A Hmostaseologie of the Normenausschuss Medizin (NaMed) (Medical Standards Committee) of DIN and has been compiled in accordance with the Technical Report procedure. Attention is drawn to the possibility that some elements of this document may be the subject of patent rights. DIN shall not be held r

11、esponsible for identifying any or all such patent rights. The number of thrombocytes (also commonly referred to as “platelets”) and platelet function play a crucial part in haemostasis, i.e. in the closure of damaged blood vessels. In addition, platelets are important components in the pathogenesis

12、of arterial thrombosis. Because of the epidemiological significance of vascular occlusion, platelet function inhibitors are among the most commonly used drugs. Platelet function diagnostics (also known as “platelet function testing”) aims to measure reduced, normal or increased platelet function. Ke

13、y diagnostic objectives include: demonstration or exclusion of platelet dysfunction in the form of bleeding diathesis or increased aggregation; demonstration or exclusion of disease-induced or drug-induced platelet dysfunction, particularly in the case of liver, kidney and bone marrow diseases. Exam

14、ples of substances or drugs showing platelet inhibition as a side effect include non-steroidal anti-inflammatory drugs, certain psychotropic medications, antihypertensives, antibiotics and a variety of dietary supplements; monitoring the correct activity of platelet function inhibitors; functional a

15、ssessment of VWF as an essential component of primary haemostasis. This following methods of assessing platelet function are described in this document: light transmission aggregometry (Borns method) (LTA); impedance aggregometry (Cardinals method); multiple electrode aggregometry (MEA); PFA systems

16、 (PFA-100, PFA-200). There is a lack of standardization among the different methods used to assess platelet function. Results, reference ranges or therapeutic ranges should have been determined using essentially the same pre-analytical and analytical methods. In particular, findings (study data, cli

17、nical experience, recommendations from working groups, guidelines) that were obtained using a common measurement principle shall not be transferred between different types of instrument without prior testing. Established reagent-instrument combinations should be used for the analysis. DIN SPEC 58961

18、2013-04 4 1 Scope The objective of this report is to describe methods of analysing platelet function in clinical laboratories. As far as possible, all aspects of pre-analytical testing, sample handling, reagent preparation, test implementation and clinical applications will be discussed insofar as

19、these are known. Methods that are primarily used for research purposes or that are never, or only rarely, used in clinical laboratories will not be addressed. 2 Symbols and abbreviations Table 1 Symbols and abbreviations ADP Adenosine 5-diphosphate ArA Arachidonic acid ASA Acetylsalicylic acid (also

20、 referred to simply as “aspirin”) AUC Area under the (aggregation) curve BAPA Benzylsulfonyl-D-Arg-Pro-4-amidinobenzylamide cAMP Cyclic adenosine 3, 5-monophosphate DDAVP 1-desamino-8-D-arginine vasopressin EIA Electrical impedance aggregometry EPI Epinephrine GPlb receptor Platelet glycoprotein Ib

21、receptor GP-IIb/IIIa receptor Platelet glycoprotein IIb/IIIa receptor COL Collagen LTA Light transmission aggregometry (Borns method) MEA Multiple electrode aggregometry NSAIDs Non-steroidal anti-inflammatory drugs P2Y12 receptor A platelet ADP receptor P2Y1 receptor A platelet ADP receptor PAR1 rec

22、eptor A platelet receptor PFA Platelet function analyser PGE1 Prostaglandin E1 PPP Platelet-poor plasma PRP Platelet-rich plasma TRAP-6 Thrombin-receptor activator for peptide 6 VC Coefficient of variation VWF von Willebrand factor VWD von Willebrand disease (also: “acquired von Willebrand syndrome”

23、) CT Closure time DIN SPEC 58961:2013-04 5 3 Pre-analytical procedures in platelet function testing 3.1 Patient preparation in platelet function testing Platelet function testing can be influenced by a variety of endogenous (patient-derived) factors and exogenous factors (blood extraction, transport

24、 storage, sample preparation). Depending on the medical issue being addressed, the following points shall be clarified before taking a blood sample for platelet function testing purposes: bleeding diathesis in patients medical history and family history; pre-existing conditions, particularly diabet

25、es mellitus, cerebrovascular diseases, renal insufficiency, myeloproliferative disorders; record of current medication, particularly platelet aggregation inhibitors, anticoagulants, NSAIDs, phosphodiesterase inhibitors, desmopressin, antibiotics, chemotherapeutic agents and plasma expanders; also wh

26、ether patient is taking naturopathic products or is receiving radiocontrast agents; consumption of alcohol and/or nicotine; when assessing the effects of platelet aggregation inhibitors, drugs that can interact detrimentally with platelet inhibitors shall be taken into account (example: proton pump

27、inhibitors when taking clopidogrel). Where possible, the patient should avoid sports activities in the 24 hours prior to a blood sample being taken. Ergometric tests (e.g. on a cycle ergometer) shall not be performed before blood collection, as sporting activity can lead to an increase in platelet a

28、ggregation induced by adenosine 5-diphosphate (ADP) and epinephrine (EPI) and to an increase in the von Willebrand factor (VWF) 1. The patient shall not consume alcohol or high-fat meals prior to blood collection. Unless the platelet-inhibiting effect of medication is being investigated, the patient

29、 shall for a period of 14 days refrain from taking medication that impairs platelet function (e.g. NSAIDs) insofar as this is clinically acceptable 2. 3.2 Blood extraction Blood is taken from a vein with the patient sitting or lying down. The tourniquet pressure should be between the systolic and di

30、astolic pressures and is released after the vein has been punctured successfully. A 19 gauge to 21 gauge puncture needle shall be used 2. Both butterfly-mounted and straight needles can be used 3. Strong negative pressure should be avoided when drawing blood so as not to activate platelet function o

31、r cause haemolysis. Blood extraction systems suitable for platelet diagnostics shall be used. If possible, the contents of the first collection tube should not be used for platelet function testing. Partially filled, coagulated or haemolytic samples shall be discarded. The type of anticoagulant used

32、 in the blood collection tube will depend on the particular method of platelet function testing to be used (light transmission aggregometry, impedance aggregometry, PFA systems). 3.2.1 Light transmission aggregometry (Borns method) (LTA) Most of the results from Borns method of light transmission ag

33、gregometry have been obtained using blood anticoagulated with citrate. Blood is extracted using collection tubes containing a sterile 0,109 M (3,2 %) solution of trisodium citrate (Na3C6H5O7) in a volume ratio of 1 part anticoagulant to 9 parts venous blood. Immediately after extraction, the blood a

34、nd anticoagulant are mixed well by gently inverting the tube 3 to 6 times without foaming. However, the use of citrate as an anticoagulant is not ideal, as platelet aggregation is impaired due to the unphysiologically low calcium concentration resulting, for example, in enhanced platelet response to

35、 ADP 4, 5. As citrate does not fully inhibit thrombin formation in extracted blood and as platelet vitality is reduced in blood anticoagulated with citrate, measurements of platelet function in blood containing citrate anticoagulant shall be conducted within 4 hours to prevent measurement errors. Th

36、e limitations associated with citrate as an anticoagulant have led to the study of other anticoagulating agents for use in platelet function testing. Hirudin and benzylsulfonyl-D-Arg-Pro-4-amidinobenzylamide (BAPA), in particular, exhibit beneficial blood anticoagulation properties when compared wit

37、h citrate 6, 7. Recombinant hirudin does not adversely affect the calcium concentration in the blood sample. However, even in high concentrations, hirudin is unable to fully inhibit thrombin formation after blood extraction so that thrombin-induced platelet activation should be expected in stored sa

38、mples 6. DIN SPEC 58961:2013-04 6 3.2.2 Multiple electrode aggregometry (MEA) and impedance aggregometry If MEA is carried out using the Multiplate system from Dynabyte (Munich)1), blood is collected into tubes containing hirudin to yield a volume ratio of 1 part hirudin (15 g/ml to 35 g/ml) to 9 pa

39、rts venous blood 2. For impedance aggregometry, blood anticoagulated with citrate is extracted using collection tubes containing 0,109 M citrate (3,2 %, see 4.2.1) 2. 3.2.3 Platelet function analyser (PFA systems) The blood should be extracted directly into a plastic or siliconized-glass evacuated t

40、ube or into a syringe containing 3,8 % (0,129 M) or 3,2 % (0,109 M) buffered sodium citrate (1 part anticoagulant to 9 parts blood). The use of 3,8 % buffered sodium citrate is preferred. The use of unbuffered sodium citrate is not recommended. 3.3 Determination of platelet count and packed cell vol

41、ume To assess platelet function, platelet count and the packed cell volume (also: “haematocrit”) should be known. The platelet count shall be at least 100/nl if light transmission aggregometry (Borns method) or impedance aggregometry is used. A lower platelet count is acceptable in clinical diagnost

42、ics, though only qualitative conclusions can be drawn in that case. According to the usage instructions for the PFA systems, closure times become longer at a platelet count of less than 100/nl and generally become pathologically extended at a platelet count below 50/nl. Abnormal PFA closure times ca

43、n also be expected at packed cell volumes of below 30 % and above 50 % 2, 8. 3.4 Transporting samples Transport should be carried out at room temperature, with acceptable transport times varying depending on the method. Samples should be subjected to the minimum possible agitation. Transport by cour

44、ier service is desirable. Pneumatic tube transport systems may be used if, in each individual case, comparative measurements have demonstrated that platelet function is not impaired by passage through the pneumatic tube. Samples shall not be cooled during transport, as low temperatures can modify pl

45、atelet function. 3.5 Sample preparation 3.5.1 Light transmission aggregometry (Borns method) (LTA) After extraction, the blood sample shall be left at room temperature (preferably between 20 C and 25 C) for between 30 minutes and 60 minutes. The sample is then centrifuged at 170 g for 15 minutes at

46、room temperature without application of the brake to prepare platelet-rich plasma (PRP). Alternative centrifugation protocols are possible (100 g to 200 g for 5 minutes to 15 minutes) 2. The PRP is transferred into plastic tubes using plastic pipettes. The platelet count is determined in a cell coun

47、ter and the result recorded. Diluting to a specified platelet count in the PRP is unfavourable at concentrations above 800/nl and is no longer recommended. PPP is prepared by centrifugation of the blood sample remaining after removal of PRP at 1 500 g to 2 000 g for between 10 minutes and 15 minutes

48、 at room temperature. Alternative centrifugation protocols are possible using a PDQTM2) microprocessor-controlled high-speed bench centrifuge designed for rapid blood separation (rotor at an angle of 30 and 4 440 g for 120 s). The platelet count in the PPP shall be less than 10/nl as demonstrated and documented by measurements in a cell counter. To prevent changes in 1) The Multiplatesystem from Dynabyte-Informationssysteme GmbH (Munich) is an example of a suitable commercially available