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WHO DIL LAB 99 1 Rev 2,World Health Organization, This document is not a formal publication of the World Health Organization WHO but all rights are. reserved by the Organization The document may however be freely reviewed abstracted reproduced. or translated in part or in whole but not for sale or for use in conjunction with commercial purposes. The views expressed by named authors are solely the responsibility of those authors. WHO DIL LAB 99 1 Rev 2,Original ENGLISH,Distr GENERAL. USE OF ANTICOAGULANTS IN DIAGNOSTIC,LABORATORY INVESTIGATIONS. Stability of blood plasma and serum samples,Contributors. G Banfi Milan Italy H Kitta Usingen Germany,K Bauer Vienna Austria D Klahr Tuttlingen Germany.
W Brand N mbrecht Germany D Kolpe N mbrecht Elsenroth Germany. M Buchberger Kremsm nster Austria J Kukuk Limburg Germany. A Deom Geneva Switzerland T Kunert Latus Leuven Belgium. W Ehret Augsburg Germany M Lammers Marburg Germany. W D Engel Mannheim Germany E A Lepp nen Helsinki Finland. F da Fonseca Wollheim Berlin Germany P Mikulcik Fernwald Germany. C G Fraser Dundee Scotland S Narayanan New York USA. V J Friemert Deisenhofen Germany M Neumaier Hamburg Germany. S Golf Giessen Germany M A Pe a Amaral Gomes Lisbon Portugal. H Gross Hanau Germany R Probst Munich Germany, W G Guder M nchen Germany Y Schmitt Darmstadt Germany. G Gunzer Clare Ireland O Sonntag Neckargem nd Germany. P Hagemann Z rich Switzerland G T pfer G rlitz Germany. W Heil Wuppertal Germany R Weisheit Penzberg Germany. J Henny Nancy France H Wisser Stuttgart Germany, R Hinzmann Krefeld Germany B Zawta Mannheim Germany. P Hyltoft Persen Odense Denmark R Zinck Mannheim Germany. G Hoffmann Grafrath Germany, A Kallner Stockholm Sweden Member of working group. A Karallus Heidelberg Germany Chairman of working group. WHO DIL LAB 99 1 Rev 2,WHO DIL LAB 99 1 Rev 2, The WHO document Use of Anticoagulants in Diagnostic Laboratory Investigations. WHO DIL LAB 99 1 Rev 1 received a surprising resonance and experts around the world provided. many additional observations This information has been included in the 2nd revision of the document. The document provides an extensive summary of observations on the effects of anticoagulants in. blood plasma and serum Information on the effects of haemolysis hyperbilirubinaemia and. hyperlipoproteinaemia on measurement procedures has been added. WHO is grateful for the efforts made by the group of experts in collecting all the information necessary. for this revised version,Geneva 15 January 2002,WHO DIL LAB 99 1 Rev 2.
1 Serum Plasma or Whole Blood Which Anticoagulants to Use 5. 1 1 Definitions 5,1 1 1 Whole blood 5,1 1 2 Plasma 5. 1 1 3 Serum 5,1 1 4 Anticoagulants 5,1 2 Plasma or serum 6. 1 2 1 Advantages of using plasma 6,1 2 2 Disadvantages of plasma over serum 6. 1 2 3 Analytical samples in the serological diagnosis of infectious diseases 7. 1 3 Recommendations 7,1 3 1 Sample collection and transport time 7. 1 3 2 Centrifugation 7,1 3 3 Storage 8,1 3 4 Evaluation of new analytical procedures 8.
2 The Optimal Sample Volume 8,2 1 Definition 8,2 2 Recommendations 9. 2 2 1 Measures which can help to reduce the required blood volume 9. 2 2 2 Documentation 9,3 Analyte Stability in Sample Matrix 9. 3 1 Stability and Instability 10, 3 2 Quality assurance of the time delay during the pre analytical phase 10. 3 2 1 Transport time 10,3 2 2 Pre analytical time in the laboratory 10. 3 2 3 Documentation 10, 3 2 4 Actions to be taken when the maximum permissible pre analytical times are.
exceeded 10,4 The Haemolytic Icteric and Lipaemic Sample 11. 4 1 Definition of a clinically relevant interference 11. 4 2 General recommendations 11,4 2 1 Documentation of interferences 11. 4 2 2 Detection of a potentially interfering property and handling of sample. and request 12,4 2 3 Reporting results 12, 4 3 The haemolytic sample and the effect of therapeutic haemoglobin derivatives 12. 4 3 1 Haemolysis 12, 4 3 2 Haemoglobin based oxygen carriers used as blood substitutes 12. WHO DIL LAB 99 1 Rev 2, 4 3 3 Detection and measurement of haemoglobin in serum or plasma 13.
4 3 4 Distinction between in vivo haemolysis and in vitro haemolysis 13. 4 3 5 Mechanisms of interference by haemolysis 13, 4 3 6 Means to avoid haemolysis and its interferences 14. 4 3 7 Reaction upon the receipt of haemolytic samples 14. 4 4 The Lipaemic Sample 15,4 4 1 Definition 15,4 4 2 Causes of lipaemia turbidity 15. 4 4 3 Identification and quantification of lipaemia 15. 4 4 4 Mechanisms of the interference by lipaemia on analytical methods 15. 4 4 5 Means to avoid lipaemia and interferences caused by turbidity 16. 4 4 6 Recommendation 17,4 4 7 Test of interference by lipaemia 17. 4 5 The icteric sample 17, 4 5 1 Appearance of different bilirubin species 17. 4 5 2 Mechanisms of bilirubin interference 17, 4 5 3 Detection and documentation of increased bilirubin concentrations in clinical.
samples 18,4 5 4 Prevention of bilirubin interference 18. 5 Samples and Stability of Analytes 20,5 1 Blood 21. 5 2 Urine 46,5 3 Cerebrospinal Fluid CSF 50,6 References 51. WHO DIL LAB 99 1 Rev 2, 1 Serum Plasma or Whole Blood Which Anticoagulants to Use. It is imperative that the in vivo state of a constituent remains unchanged after withdrawal from the. body fluid of a patient to obtain a valid medical laboratory result This may not always be possible. when measuring extra cellular and cellular components of blood Platelets and coagulation factors are. activated when blood vessels are punctured and their activation continues in sample containers that do. not contain anticoagulant, Historically serum was the preferred assay material for determining extracellular concentrations of.
constituents in blood Today plasma is preferred for many but not all laboratory investigations. because the constituents in plasma are better reflecting the pathological situation of a patient than in. serum Some changes of constituents can be avoided by using anticoagulants The types and. concentrations of anticoagulants used in venous blood samples were defined in the international. standard 86 in 1996 The standardized anticoagulants are now used to prepare standardized plasma. samples for laboratory investigations throughout the world. This document summarizes the findings published in the literature and those observed by the. contributors on the use of anticoagulants The overview was prepared in collaboration with experts. from clinical diagnostic laboratories and the diagnostics industry 68 71. 1 1 Definitions,1 1 1 Whole blood, A venous arterial or capillary blood sample in which the concentrations and properties of cellular and. extra cellular constituents remain relatively unaltered when compared with their in vivo state. Anticoagulation in vitro stabilizes the constituents in a whole blood sample for a certain period of time. 1 1 2 Plasma, The virtually cell free supernatant of blood containing anticoagulant obtained after centrifugation. 1 1 3 Serum, The undiluted extracellular portion of blood after adequate coagulation is complete. 1 1 4 Anticoagulants, Additives that inhibit blood and or plasma from clotting ensuring that the constituent to be measured is. non significantly changed prior to the analytical process Anticoagulation occurs by binding calcium. ions EDTA citrate or by inhibiting thrombin activity heparinates hirudin The following solid or. liquid anticoagulants are mixed with blood immediately after sample collection. 1 1 4 1 EDTA, Salt of ethylene diamine tetraacetic acid Dipotassium K2 tripotassium K3 41 and disodium Na 2.
salts are used 86 concentrations 1 2 to 2 0 mg mL blood 4 1 to 6 8 mmol L blood based on. anhydrous EDTA,1 1 4 2 Citrate, Trisodium citrate with 0 100 to 0 136 mol L citric acid Buffered citrate with pH 5 5 to 5 6. 84 mmol L tris odium citrate with 21 mmol L citric acid Differences were noticed between 3 2 and. 3 8 v v citrate when reporting results in INR 1 145 192 210 WHO and NCCLS recommend. 0 109 mol L 3 2 citric acid The International Society for Thrombosis and Haemostasis ISTH. recommends the use of Hepes buffered citrate for all investigations of haemostastic functions 114. a A mixture of one part citrate with nine parts blood is recommended for coagulation tests. b One part citrate mixed with four parts blood is recommended to determine the erythrocyte. sedimentation rate 86,WHO DIL LAB 99 1 Rev 2,1 1 4 3 Heparinates. 12 to 30 IU mL of unfractionated sodium lithium or ammonium salt of heparin with a molecular mass. of 3 to 30 kD is recommended to obtain standardized heparinized plasma 86. Calcium titrated heparin at a concentration of 40 to 60 IU mL blood dry heparinisation and 8 to 12. IU mL blood liquid heparinisation is recommended for the determination of ionized calc ium 22. 1 1 4 4 Hirudin, Hirudin is an antithrombin extracted from leeches or prepared by a genetic engineering process. Hirudin inhibits thrombin by forming a 1 1 hirudin thrombin complex Hirudin is used at a. concentration of 10 mg L 40, The colour codes of anticoagulants described in ISO DIS 6710 are. EDTA lavender red,citrate 9 1 light blue green,citrate 4 1 black mauve.
heparinate green orange,no additives for serum red white 86. 1 2 Plasma or serum,1 2 1 Advantages of using plasma. The following aspects support the preferential use of plasma versus serum in laboratory medicine. Time saving Plasma samples can be centrifuged directly after sample collection unlike serum in. which coagulation is completed after 30 minutes, Higher yield 15 to 20 more in volume of plasma than of serum can be isolated from the same. volume of blood, Prevention of coagulation induced interferences Coagulation in primary and secondary tubes that. were already centrifuged may block suction needles of the analyzers when serum tubes are used. this is prevented by using anticoagulants, Prevention of coagulation induced interferences The coagulation process changes the concentrations.
of numerous constituents of the extra cellular fluid beyond their maximum allowable limit 70. 202 The changes are induced by the following mechanisms. a Increase in the concentrations of platelet components in serum as compared to plasma e g. potassium phosphate magnesium aspartate aminotransferase lactate dehydrogenase. serotonin neurone specific enolase zinc Release of amide NH3 from fibrinogen induced by. action of clotting factor XIII, b Decrease in the concentration of constituents in serum as a result of cellular metabolism and. the coagulation process glucose total protein platelets. c Activation of the cell lysis of erythrocytes and leukocytes in non coagulated blood cell free. haemoglobin cytokines receptors, Certain constituents should only be measured in plasma e g neurone specific enolase serotonin. ammonia to obtain clinically relevant results,1 2 2 Disadvantages of plasma over serum. The addition of anticoagulants may interfere with certain analytical methods or change the. concentration of the constituents to be measured,a Contamination with cations NH4 Li Na K. WHO DIL LAB 99 1 Rev 2, b Assay interference caused by metals complexing with EDTA and citrate e g inhibition of alkaline.
phosphatase activity by zinc binding inhibition of metallo proteinases inhibition of metal. dependent cell activation in function tests binding of calcium ionized to heparin 22. c Interference by fibrinogen in heterogeneous immunoassays 202. d Inhibition of metabolic or catalytic reactions by heparin e g Taq polymerase in the polymerase. chain reaction PCR 137, e Interference in the distribution of ions between the intracellular and extracellular space e g. Cl NH4 by EDTA citrate 70, f Serum electrophoresis can be performed only after pre treatment to induce coagulation in plasma. 1 2 3 Analytical samples in the serological diagnosis of infectious diseases. A variety of methods are used for serological diagnosis of infectious diseases They include immuno. diffusion immuno precipitation counter immuno electrophoresis agglutination of bacteria. haemagglutination and agglutination inhibition particle enhanced agglutination complement fixation. indirect immuno fluorescence IFA enzyme linked immunoassay ELISA radio immunoassay. RIA neutralisation of toxins or virus activity immunoblot Western blot and others. In general serum is used for the serological diagnosis of infectious diseases serum must be used for. certain immunological techniques such as complement fixation or bacterial agglutination tests for. other tests including some haemagglutination tests ELISAs or immunoblots either serum or plasma. may be used,1 3 Recommendations, Table 5 1 indicates materials that are recommended for a specific test The table also contains. information on the utility of other sample materials as long as the results measured by that method do. not exceed the maximum allowable deviation of measurement 152 as defined by the biological. variation 153 A maximum deviation of 10 is acceptable for a constituent if it is not included in the. 1 3 1 Sample collection and transport time, The following sequence for filling tubes with blood from a patient is recommended to avoid. contamination 70,blood for blood culture, serum avoid serum as first tube when electrolytes shall be measured 116.
citrate heparinate EDTA containing tubes, tubes containing additional stabilizers e g glycolytic inhibitors. Only the recommended quantity of anticoagulant should be added wherever required to avoid errors in. Tilt the tube repeatedly do not shake and avoid foaming immediately after filling to mix the sample. thoroughly with the anticoagulant Leave the containers at room temperature for at least 30 minutes to. separate serum from blood cells in blood that was taken from non anticoagulated patients This period. is shorter when coagulation has been activated Leave the sample at room temperature no longer than. the period indicated in the table see 5 1 66,1 3 2 Centrifugation. Blood cells are rapidly separated from plasma serum by centrifugation at increased relative centrifugal. force rcf Rcf and rotations per minute rpm are calculated using the rotating radius r the distance. between the axis of rotation and the base of the container in mm by the following equation. rcf 1 118 x r rpm 1000 2,WHO DIL LAB 99 1 Rev 2, Centrifuge blood containers in 90 swing out rotors that the sediment surface forms a right angle with. the container wall This helps to prevent contact between the sampling needle and the surface of the. cell layer or separating gel in the tube when the centrifuged blood containers are directly transferred to. an autoanalyzer for analysis,1 3 2 1 Plasma, Centrifuge the anticoagulated blood citrated EDTA or heparinized blood for at least 15 minutes at. 2000 to 3000 g to obtain cell free plasma,1 3 2 2 Serum.
When plasma coagulation is complete centrifuge the sample for at least 10 minutes at a minimum. speed of 1500 g, When separating serum or plasma the temperature should not drop below 15 C or exceed 24 C. 1 3 3 Storage, Non centrifuged samples should be stored at room temperature for the time specified in the. recommendations for stability see table 5 1 66 After centrifugation the serum or plasma should be. analyzed within the time as recommended for whole blood if the sample is stored without using a. separating gel or a filter separator in primary tubes When the sample shall be refrigerated or frozen for. preservation blood cells must first be separated from serum or plasma Do not freeze whole blood. samples before or after centrifugation even when polymer separating gels are used. 1 3 4 Evaluation of new analytical procedures, Before using a new reagent or method examine the suitability of the procedure by comparing the. results of at least 20 blood samples with normal and 20 with pathological concentrations of the. constituent to be measured The criteria for biological and clinical interpretation reference intervals. clinical decision limits may have to be changed if the mean of the difference between the samples. tested deviates by more than the maximum deviation allowed 152 alternatively by more than 10. 2 The Optimal Sample Volume, The progress in the development of laboratory analyzers has led to a reduction of the sample volume. for analysis The development however is not necessarily accompanied by an adaptation of sample. tubes and therefore often excessive sample volumes are collected Studies revealed 30 that 208 mL. blood for 42 tests is taken during an average stay of a patient in a department of internal medicine In. intensive care the total volume drawn for 125 tests was 550 mL of blood Previous publications. describe that in half of the patients who received blood transfusion more than 180 mL of blood were. taken for laboratory tests 174 Iatrogenic anaemia caused by excessive blood sampling is a well. known phenomenon in paediatrics 27 whereas iatrogenic anaemia is hardly recognized as an. important phenomenon in the acute and intensive care of adult patients The following. recommendations were made for sampling reduced blood volumes for analysis 67. 2 1 Definition, The amount of sample needed for laboratory diagnostic purposes Vol b is defined by.
1 The analytical sample volume Vol a, 2 The dead space volume of the analyser Da measured as mL plasma serum. 3 The dead space volume of the primary sample tube Dp measured as mL blood. 4 The dead space volume of secondary sample tubes Ds measured as mL plasma serum. 5 The amount of sample needed for number N repetitive analysis and additional follow up tests. 6 The plasma sample yield according to the respective haematocrit. WHO DIL LAB 99 1 Rev 2, Assuming that plasma serum yield is 50 of blood volume the total blood needed can be calculated as. Vol b 2x N x Vol a Da Ds Dp,2 2 Recommendations, Assuming a haematocrit of 0 50 and a need for a repetition and follow up of laboratory tests four times. the analytical sample volume can be considered to be sufficient when plasma or serum shall be used. The following standard blood volumes are recommended for analysis using advanced analytical. systems These volumes may be sufficient in 95 of cases to provide the laboratory results as. Clinical chemistry 4 5 mL when using heparin plasma 3 4 mL. Haematology 2 3 mL EDTA blood,Coagulation tests 2 3 mL citrated blood. Immunoassays including proteins etc 1 mL whole blood for 3 4 immunoassays. Erythrocyte sedimentation rate 2 3 mL citrated blood. Blood gases capillary sampling 50 l arterial and venous sampling 1 mL heparin blood. The request form for laboratory analyses should include clear information on the required sample. volumes and tubes Tubes of uniform size for instance 4 5 mL tubes with different filling volumes. should be used The length of the tubes should be at least four times the tube diameter The criteria are. met by standard tubes of 13 x 75 mm diameter x length. 2 2 1 Measures which can help to reduce the required blood volume. Introduction of primary tube reading in analyzers, Deletion of sample distribution into secondary tubes.
Use of tubes with smaller diameter, Use of analyzers requiring a smaller analytical sample volume. Storage of samples in primary tubes using separators for plasma or serum. Use of plasma instead of serum,2 2 2 Documentation. 1 Any method description should include the required analytical sample volume. 2 A quality manual should document the requested sample volumes and their handling procedure. 3 The manual should describe the procedures how to handle patient samples that have an insufficient. sample volume,3 Analyte Stability in Sample Matrix. The aim of a quantitative laboratory investigation is to determine the concentration or activity of a. diagnostically relevant analyte in a body fluid in order to provide information on the clinical situation. of a patient This implies that the composition of the samples for analysis must not change during the. pre analytical phase sampling transportation storage sample preparation 70. WHO DIL LAB 99 1 Rev 2,3 1 Stability and Instability. Stability is the capability of a sample material to retain the initial property of a measured constituent for. a period of time within specified limits when the sample is stored under defined conditions 87. The measure of the instability is described as an absolute difference as a quotient or as a percentage. deviation of results obtained from measurement at time 0 and after a given period of time. The transportation of whole blood for 3 to 4 hours at room temperature rises the concentration of. potassium from 4 2 mmol L to 4 6 mmol L,Absolute difference 0 4 mmol L.
Quotient 1 095,Percent deviation 9 5, The maximum permissible instability is the deviation of a result that corresponds to the maximum. permissible relative imprecision of the measurement This was defined as 1 12th of the biological. reference interval 152 The deviation should be smaller than half of the total error derived from the. sum of biological and technical variability 153 The stability of a blood sample during the pre. analytical phase is defined by the temperature the mechanical load in addition to other factors As time. has also a major influence the stability is stated as the maximum permissible storage time under. defined conditions, The maximum permissible storage time is the period of time at which the stability requirement of 95. of the samples is met This is a minimum requirement since under pathological conditions the. stability of an constituent in the sample can be considerably reduced See examples in Table 5 1. The storage time is stated in suitable units of time days hours minutes A clear distinction must be. made between the storage of the primary sample blood urine cerebrospinal fluid and the storage of. the analytical sample e g plasma serum sediment blood smear The storage times are adopted for. 1 Storage of the primary sample at room temperature 20 to 25 C. 2 Storage of the analytical sample at room temperature 20 to 25 C refrigerator temperature. 4 to 8 C and deep frozen 20 C, 3 2 Quality assurance of the time delay during the pre analytical phase. 3 2 1 Transport time, The transport time is the difference between the blood sampling time in general with an accuracy of at. least a quarter of an hour and the registration time of the request and or the arrival of the sample at the. laboratory The transportation time should be documented for each sample by the laboratory. 3 2 2 Pre analytical time in the laboratory, The pre analytical time in the laboratory is the difference between the time of analysis and the.
registration time of the sample When the time at the end of the analytical phase i e printing time of. the result is noted the analysis time stated in the description of the method must be subtracted. 3 2 3 Documentation, It is recommended to state the sampling time and the arrival time of the sample in the laboratory in the. report for the documentation of the transport time. 3 2 4 Actions to be taken when the maximum permissible pre analytical times are exceeded. A medically relevant change of the results must be considered when the maximum permissible. transport and pre analytical time of the sample was exceeded The laboratory has the responsibility to. mark the results of such samples with a note in the report or to refuse to carry out the test The latter. WHO DIL LAB 99 1 Rev 2, decision is advisable when medical conclusions may be derived from the result that may be. detrimental to the patient The following example illustrates the problem. An EDTA blood sample shows a rise in monocyte number from 4 to 10 after four hours of. storage measured by an automatic cell counter system When this results is reported without. comment it could lead to an erroneous medical diagnosis that the patient suffers from a viral. infection Therefore the clinician should be informed with a comment or a refusal such as. Comment The monocyte count may give incorrectly high values with the method used in our. laboratory when EDTA blood is stored more than 2 hours A control in the smear resulted in. normal monocyte counts, Refusal The maximum permissible transportation time was exceeded Therefore the monocyte. results are not stated because they cannot correctly be determined For the determination of. correct monocyte counts a transportation time of maximally two hours is acceptable. 4 The Haemolytic Icteric and Lipaemic Sample, Medical laboratory tests are affected by endogenous and exogenous factors in the sample matrix. Certain potentially interfering factors may be recognized by a coloured appearance of the sample. whereas other factors e g drugs are detected only by additional information and or direct analysis. Reference books provide useful information on drug interferences in laboratory analysis 178 194. 214 Publications of standard setting organizations describe the methodology and statistical methods. for the recognition and quantitative estimation of interferences in clinical chemical investigations 52. It is difficult to predict the effects of haemolysis turbidity lipaemia and bilirubin icterus especially. when reagents and analytical systems undergo modification 58 59 This document provides. information that the laboratory can consider appropriate actions to ensure that the results of. measurement are clinically relevant, 4 1 Definition of a clinically relevant interference.
The maximal allowable deviation bias is expressed in deviation of the result without interference. as determined by a reference method A clinically relevant bias should be considered if the change of. the result caused by the interfering substance is more than the maximal allowable deviation of the. analytical procedure 152 The bias usually amounts to 1 12 which is about 8 of the reference. Data on the biological variability were published to define the medical needs The desirable bias B. derived from intra individual CVw and inter individual CVb variation was established for 316. analytes 153, A result for plasma creatinine of 125 mol L 1 41 mg dL was measured in an icteric sample. by a routine method whereas a creatinine concentration of 90 mol L 1 02 mg dL was. measured in the same sample by a reference method For creatinine the maximum allowable. deviation amounts to 9 158 the specification for B to 3 4 153 The result deviates by. 35 mol L which is 39 from the expected value Both criteria confirm that. hyperbilirubinaemia is a clinically relevant interference when creatinine is measured using the. routine method established in the laboratory,4 2 General recommendations. 4 2 1 Documentation of interferences, Documentation of method Each clinical laboratory should specify the constituents in the quality. manual that are affected by any of the following properties of the sample The limits beyond which the. analysis shall not be performed should be stated for each method that is subject to an interference The. WHO DIL LAB 99 1 Rev 2, European Directive for In Vitro Diagnostics IVD states that providers of reagents must define the. appropriate limiting conditions 42 The procedure for the detection of interfering properties as well as. actions that should be taken with the sample should be documented in the quality manual. 4 2 2 Detection of a potentially interfering property and handling of sample and request. Each sample must be visually examined immediately after arrival or in case of blood samples after. centrifugation and the potential interfering property recorded in the laboratory journal and report When. no visible interference is observed it should be registered in the list by the notation appearance. unremarkable, The requests should be reviewed to identify analytes that could be affected by the observed interference.
in the sample Analytes that are not affected by the interference in the sample are measured like in. samples that contain no interference using the routine method of analysis A sample that may be. expectedly affected by an identified interference must be pre treated to eliminate the interference. before measurement is made alternatively a measurement method may be used that is not subject to the. interference The analysis should not be made when a clinically relevant bias is expected or if the. interference cannot be eliminated or circumvented by an appropriate alternative method. 4 2 3 Reporting results, Each report should include a notation characterising the samples appearance The observation should. be documented for each sample e g haemolytic icteric opalescent turbid or lipaemic if a. relevant colour or turbidity was identified, The report should indicate that the analysis was made despite a remarkable appearance of an. interference in the sample The report should also indicate when the sample was pre treated prior to the. analysis If the interference in a sample cannot be eliminated for a subsequent analysis the text. impaired by should replace the report of the result. 4 3 The haemolytic sample and the effect of therapeutic haemoglobin derivatives. 4 3 1 Haemolysis, Haemolysis is defined as the release of intracellular components of erythrocytes and other blood cells. into the extracellular space of blood 65 Haemolysis can occur in vivo e g through a transfusion. reaction or during malaria parasite infection affecting the invaded erythrocytes and in vitro during all. steps of the pre analytical phase sampling sample transport and storage. Haemolysis is caused by biochemical immunological physical and chemical mechanisms 18 65. During blood transfusion complement dependent haemolysis may be caused by antibodies reacting. with the major blood group antigens Physical haemolysis is caused by destruction of erythrocytes by. hypotonicity e g dilution of blood with hypotonic solution as well as decreased vacuum or. increased pressure Mechanical haemolysis may occur during the flow of blood through medical. devices e g catheters heart valves in vivo during inadequate centrifugation as well as at elevated. temperature in vitro Contaminating substances may also cause in vitro haemolysis Finally detergents. residual cleaning agents and disinfectants and other contaminating substances may cause haemolysis. After the separation of blood cells haemolysis may be visible by the red colour of serum or plasma. The sample may concomitantly be contaminated by constituents of other blood cells leukocytes and. platelets For example cell breakdown may result in changes in blood of patients with leukaemia the. disintegration of platelets during coagulation results in higher concentrations of intracellular platelet. constituents in serum 70 On the other hand the intracellular components of erythrocytes are also. released into plasma without a concomitant increase in haemoglobin concentration during storage of. whole blood in refrigerators, 4 3 2 Haemoglobin based oxygen carriers used as blood substitutes. Therapeutic haemoglobin derivatives so called HbOC haemoglobin based oxygen carriers were. recently developed as blood substitutes The substitutes occur at concentrations of up to 50 g L in. WHO DIL LAB 99 1 Rev 2, plasma of patients under blood substitute treatment Plasma or serum containing blood substitutes has.
a strong red colour 24 92 211, 4 3 3 Detection and measurement of haemoglobin in serum or plasma. 4 3 3 1 Visual detection, At extracellular haemoglobin concentrations above 300 mg L 18 8 mmol L haemolysis is detectable. by the red colour of serum or plasma Samples with therapeutic haemoglobin derivatives in. therapeutically effective concentration are always intensely red coloured. 4 3 3 2 Spectrophotometric detection, Some analytical systems measure the extent of haemolysis by comparing the absorption of samples at. two wavelengths 61 The absorption spectrum of the haemoglobin derived oxygen carriers used as. blood substitutes does not differ substantially from that of natural haemoglobin. 4 3 3 3 Analytical measurement, Haemoglobin in plasma or serum is measured at concentrations that are below the concentration visible. to the human eye 13 110 188, 4 3 4 Distinction between in vivo haemolysis and in vitro haemolysis.
In vivo haemolysis may be distinguished from in vitro haemolysis by comparing a haemolytic sample. of a patient with other samples from the same patient arriving at the same time. 4 3 4 1 In vivo haemolysis, Free haemoglobin in vivo rapidly binds to haptoglobin and the complex is eliminated from the. circulating blood as in haemolytic anaemia Consequently haptoglobin is reduced during intra vasal. haemolytic process The measurement of low concentration of haptoglobin thus permits an imperative. assessment of haemolysis exceptions are inborn haptoglobin deficiency and newborn children 199. Likewise the measurement of haemopexin and or methaemoglobin albumin was used to characterize. in vivo haemolysis 199, A rise in concentration of indirect bilirubin and reticulocyte counts is a typical sign of in vivo. haemolysis which in turn leads to increased erythropoesis Other consequences of in vivo haemolysis. such as a change in the LDH iso enzyme pattern seem less suitable for the identification of haemolysis. because of their low diagnostic sensitivity and specificity. 4 3 4 2 In vitro haemolysis, After in vitro haemolysis all constituents of erythrocytes including potassium concentration lactate. dehydrogenase and aspartate aminotransferase activities increase in addition to the concentration of. free haemoglobin in plasma or serum 208 In contrast haptoglobin concentration in plasma serum of. haemolytic samples remains unchanged Certain immunological methods differ in their ability to. distinguish haemoglobin haptoglobin complexes from free haptoglobin 199. 4 3 4 3 Identification of haemoglobin derived oxygen carriers. Therapeutic haemoglobin derivatives yield a visible haemoglobin concentration within the range of 10. 50 g L The absorption spectrum of haemoglobin derived oxygen carriers is not distinguishable from. that of haemoglobin 24 92 211 However haemoglobin concentrations of this magnitude rarely. occur in vivo therefore the use of therapeutic haemoglobin derivatives must be suspected at this. plasma haemoglobin concentration Haptoglobin cannot be used for discrimination since the oxygen. carriers form complexes with haptoglobin only slowly. 4 3 5 Mechanisms of interference by haemolysis, Haemolysis in vivo or in vitro can cause an apparent decrease or increase of results A variety of. mechanisms are contributing to these effects some of which are summarized below. 4 3 5 1 Rise of intracellular constituents in the extra cellular space. Cell constituents with an intracellular concentration 10 times higher than the extra cellular. concentration will increase in plasma serum during haemolysis e g potassium lactate dehydrogenase.

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