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Unformatted text preview: HAEMOSTASIS & THROMBOSIS ATTACHMENT COURSE 28/10/2002 – 7/12/2002 Haemophilia Centre & Haemostasis Haemophilia Unit, Royal Free Hospital Unit, RFH Organisational Set-up Director: Professor Christine A Lee Consultant: Dr Simon Brown Senior Lecturer: Dr David Perry Service Manager: Angus McCraw 1 . Week:28/10/02 – 1/11/02 (Morning) st Attended the morning lectures with the MRCPath Attended students students Topics included: Introduction to the Coagulation Laboratory-Sarah Introduction Brooks Brooks Newer Techniques in Haemostasis-Anne Riddell Prothrombotic State & Lab Investigation-Dr David Prothrombotic Perry Perry VWF and VWD-Dr Simon Brown Laboratory Set-up 1) Routine Haemostasis Laboratory: Staffs: i) Pura Lawler: i) ii) Sunila: iii) Ruth: iv) Marleve Dean: v) Anne Harvey: v) vi) Jeremy: vii) Adam: MLSO 2(III) MLSO 1(?) Locum MLSO Trainee MLSO Laboratory Clerk Locum MLA Locum MLA 1) Routine Haemostasis Laboratory: Tests done: a) PT/APTT a) b) TT/RT c) FIB-C d) D-Dimer e) Factors Assays: FVII, FVIII, FIX, FXI f) Inhibitor Screens g) Mixing Tests h) Nijmegan Inhibitor Assays i) Others: Fletcher, Fitzgerald, PF4, etc. i) Reception Counter Adam, Pura, Lim, Marleve Sample Reception i) i) Samples must arrive in the laboratory in sealed plastic bags, which have separate pockets for the sample and request form. form. ii) Samples are bar-coded, checked for suitability against the ii) tests requested, and verified against the request forms. tests iii) Unsuitable samples and requests with discrepancies are iii) noted and informed to the ward. noted iv) The particulars of each request are then keyed into the LIS, iv) first time requests that are not previously tested are stamped “PU” (previously untested) on the request forms and are required to be assayed in duplicate. and v) The various work-ups requests, with the plasmas separated v) and aliquoted are kept in the deep-freezers in the various sections. various vi) Platelet studies samples are sent straight to the platelet vi) laboratory on every Tues. & Wednesdays. laboratory Sample Reception vii) For Bethesda assay, previous levels are vii) recorded and monitored in a card for each patient. Fume Hood ACL Futura Coagulation Analyzer Bench-top fridge Good Laboratory Practice in Coagulation Laboratory 1)Reagent reconstitution: i)IL Lyophilised silica for APTT- 9mls dH2O, shake i)IL vigorously, leave 30’ RT before use. vigorously, ii)IL PT-HS Plus- pour entire diluent, mix gently, leave ii)IL 30’RT before use. 30’RT iii)NP-1.0ml dH2O, leave 30’RT before use. 2)Proper handling of reagents, NP and samples: i)Keep all reconstituted reagents & sera at 4C & cover lid i)Keep when not in use. DO NOT leave them unlided on the analyzer! analyzer! ii)All frozen sera & reagents must be thawed at 37C in ii)All waterbath before analysis. waterbath iii) Do not analyze lysed, clotted, underfilled/overfilled iii) samples. samples. Fridge, Hood, Refrigerated centrifuge, ACL 1000 The Nijmegan Modified Bethesda Assay for FVIII:C inhibitors Buffering the assay system with 0.1 M imidazole Buffering to pH 7.4 improves specificity and reliability. to Glyoxaline buffer is used as the pre-incubation Glyoxaline sample buffer in human Bethesda assays. sample These modifications allow better discrimination These between positive & negative samples and improve reliability. The Nijmegan Modified Bethesda Assay for FVIII:C inhibitors Interpretation and presentation of results -FVIII inhibitors may exhibit 2 forms of kinetics Type 1 Type -have a linear relationship with dilution -follows the first order kinetics with FVIII progressively neutralised until either it or the inhibitor is used up. inhibitor -completely inactivate the FVIII & there is a linear relationship when the log of the residual FVIII activity is compared to the ab concentration activity The Nijmegan Modified Bethesda Assay for FVIII:C inhibitors Type 2 Type -inhibitors do not follow a linear relationship -inhibitors -exhibit a more complex order kinetics -often give similar amounts of residual FVIII -often with different dilutions of patient’s plasma. with 2) Platelet Studies Laboratory: Tests done: a) b) c) c) d) e) Platelet aggregation tests RiCOF Assay PFA TEG test Platelet Nucleotides Bioluminescence Assay Platelet Aggregation Tests Five agonists routinely used are ADP, adrenaline, Five collagen and arachidonic acid. These are adequate to allow the major platelet function disorders to be discriminated. discriminated. If there is biphasic wave with 2uM ADP & If adrenaline, need not proceed with higher concentrations. concentrations. If patient’s platelets aggregate with 0.5mg/ml If Ristocetin (indicating possible IIB VWD or Pseudo VWD), perform spontaneous aggregation for 15 minutes. for Platelet Aggregation Tests Calculation of results: -Most convenient and recommended to measure 3 -Most mins after addition of agonist. mins Interpretation of results: -ADP at 2 uM, clearly defined primary & secondary -ADP waves can usually be seen, above 3uM, masked secondary phase; shape changed from a disc to spiky sphere. spiky -Adrenaline, patterns of response usually similar -Adrenaline, with ADP, except primary wave does not reverse nor is it so intense to mask secondary wave; no shape change. shape Platelet Aggregation Tests -With collagen, no primary wave occurs; -With response defined by duration of lag phase before onset of aggregation and its intensity. before -With ristocetin, primary wave is a measure of -With the amount of VWF present in plasma; secondary wave is due to release of endogenous substances. endogenous -With arachidonic acid, aggregation is -With monophasic & preceded by a short lag phase. phase. Platelet Aggregation Tests Precautions prior to measuring platelet Precautions aggregation – see SOP aggregation Further investigation of platelet function: -always repeat at least one occasion, if an -always abnormal aggregation pattern is observed. abnormal -in the presence of abnormal aggregation, -in further investigations include platelet nucleotides and beta-thromboglobulin; nucleotides -quantitation of membrane glycoproteins for -quantitation diagnosis of Bernard-Soulier syndrome & Thrombasthenia Thrombasthenia Ristocetin Co-factor Assay Fresh washed platelets: -prepared from ~60 ml of blood to obtain -prepared ~20ml of PRP (sufficient to run standard & 15 patients). 15 Advantages over lyophilised platelets: -cheap -cheap -wider aggregation range than lyophilised platelets platelets -disadvantage: time consuming -disadvantage: Platelet Studies Laboratory Staffs: i) Anne Riddell: i) ii) Sunila: iii)Pura Lawler: iv)Anne Harvey: v)Jeremy: vi)Adam: MLSO 3(III) MLSO 1(?) MLSO 2(III) Laboratory Clerk Locum MLA Locum MLA Madhavan, Saman, Lim, Anna 3) ELISA Laboratory: Tests done: Tests a) b) c) d) e) e) f) f) Monoclonal Free Protein S Antigen Total Protein S Protein C Ag VWF Ag CBA Factor VIII:C Levels Factor VIII:C Assay Done on IL 9000: -always include a blank, an abnormal & -always a normal control normal -assay test plasma in 3 dilutions, viz.1:5; -assay 1:10 &1:20. If values obtained deviate by >10% of each other, repeat test. by -check for parallelism, -check converging/diverging curves converging/diverging ELISA Laboratory: Staffs: i) Saman Aghighi: i) ii) Sarah Brooks: iii) Anne Harvey: iv) Jeremy: v) Adam: MLSO 2(I) MLSO 3(II) Laboratory Clerk Locum MLA Locum MLA 4) Thrombophilia Studies: Tests done: (Sample volume 4x 3mls) a) a) b) c) d) e) f) f) Lupus Anticoagulant KCT Exner Test Protein S activity Protein C activity APCr Assay Factor V Leiden/ FII 3’UTR Multiplex PCR & Restriction Analysis Lupus Anticoagulant (LA) An auto-antibody directed against , or crossreacting with, specific chemical groups that are reacting found in negatively charged phospholipids such as phosphatidyl serine and cardiolipin. phosphatidyl They are usually immediate reacting, are detected They by their effect on phospholipid-dependent coagulation tests. coagulation No one method has 100% reliability, usually > No than one test is carried out. than LA is transient in nature. Sample Preparation (LA) Double centrifugation Aliquot and store at –45C The following tests are run prior to The DRVVT/DRVVC: DRVVT/DRVVC: i)PT; ii)APTT; iii)APTT 50:50; iv)TT; i)PT; v) Fib-C Two Different LA kits are used: Manchester Reagents Russell’s Viper Manchester Venom (DRVVT & DRVVC) Venom ii) American Diagnostica Kit (DVVT & American DVVC) DVVC) If any one or both kits is/are positive, sample If is considered LA positive and proceed to do KCT screen, if >1.2, Exner ensues. do i) Lupus Anticoagulant-Calculation of results Manchester Reagents: LA screen LA -The 20NP should give a value of 45-65 s (mean 56.2) 56.2) -A test/normal ratio of 1.16 or > indicates a positive result. result. LA confirm -The 20NP should give a value of 47-56 s (mean 51.5) 51.5) -If correction is > 65%, LA positive. -If Lupus Anticoagulant-Calculation of results American Diagnostica Reagents: American LA screen LA -The 20NP should give a value of 35-45 s -Reference range for DVVT is 27-46 s -Reference LA confirm -The 20NP should give a value of 29-37s -The -Reference Ratio of DVVTest/DVVConfirm is 0.9-Reference 1.2 -A ratio of > 1.2 is considered LA positive. -A EXNER KCT Perform an Exner screen if LA is positive. Exner KCT is positive if the ratio of NP:TP Exner 8:2 to 20NP alone is 1.2 or greater. 8:2 Then, proceed to further dilutions 10:0, 9:1, Then, 5:5, 2:8 and 0:10 of 20NP to patient plasma. 5:5, Classification of the Lupus inhibitor is Classification determined by the shape of the curve by plot clotting times against dilutions. LA test: Important points to note i) ii) 20 Normal Pool used must be similarly treated as the test plasma Warfarinised samples must be mixed with equal parts of normal pooled before tested for LA.(to compensate for the effect of warfarin on FX) iii) iv) v) If LA is negative but the prolonged APTT didn’t correct well, perform KCT Heparinised samples cannot be tested for LA. Patients on warfarin cannot be tested with an Patients EXNER test. EXNER Thrombophilia Studies: Why measure Free Protein S? -Recommendations of WHO/ISTH -Free Protein S assay have higher specificity -Free & sensitivity for genetic defects causing Protein S deficiencies than total Protein S assay. assay. -Only free PS is functionally active & able to -Only bind with aPC, while the complexed form of PS is not. of -There is a great overlap in the total Protein S -There levels between normal and those with genetic defect. genetic Thrombophilia Studies: Note: If Protein C activity is normal, need not do If PC Ag. PC If free Protein S is normal, need not do total If PS. (Free PS latex particle enhanced immunoassay, IL). immunoassay, If AT activity is normal, need not do AT If Ag. Ag. Thrombophilia Studies: Staffs: i) i) ii) iii) iv) v) vi) vii) Sarah Brooks: Lesley Lanning: Saman Aghighi: Anna: Anne Harvey: Jeremy: Adam: MLSO 3(II) MLSO 2(II) MLSO 2(I) Locum MLSO Laboratory Clerk Locum MLA Locum MLA 5) DNA Studies Laboratory: Tests done: a) Mutation Studies and DNA sequencing, etc. a) Staffs: Staffs: i) Gillian Mellars: i) ii) Anne Harvey: ii) iii) Jeremy: iv) Adam: MLSO 3(I) Laboratory Clerk Locum MLA Locum MLA 6) Method Development and Multimers Laboratory: Tests done: Tests a) a) b) c) VWF Multimeric Studies Factor VIII Binding Assay Special ELISA Staffs: Staffs: i) Anne Riddell: i) ii) Jeremy: iii) Adam: MLSO 3(III) Locum MLA Locum MLA Locum Von Willebrand disease (vWD) The most common inherited bleeding The disorder caused by quantitative or qualitative defects of VWF. qualitative Prevalence of 1-2 % in the general Prevalence population. population. Different management strategies in the Different various types of vWD underlie the importance of classification. importance Classification of vWD Type 1 (partial quantitative deficiency, most Type common type) common Type 2 (qualitative defect) Type 3 (total deficiency) Based on specific structural abnormalities, Based type 2 vWD is further classified into 4 subtypes (2A, 2B, 2N, 2M) subtypes Patterns of FVIII results in vWD Notes: Type 1 is the usual heterozygous form of vWD Notes: Type 3 is very rare and presents like severe haemophilia Type Type 2 forms are the known variant forms of vWD. These are uncommon. Type FVIII:c FVIII:c Normal Normal Type 1 vWD Type 3 vWD VWF:Ag VWF:Ag VWF:Ac VWF:Ac CBA Ag:CBA Ag:CBA N N N N N N-↓ N- ↓ ↓ ↓ N Absent Absent Absent Absent - Type 2A vWD N-↓ N- N-↓ N-↓ ↓↓↓ ↑↑↑ Type 2B vWD Type N-↓ N-↓ N-↓ ↓ ↑ Type 2M vWD Type N-↓ N- N-↓ ↓↓ N-↓ N- N Type 2N vWD ↓↓↓ N N N N Initial laboratory evaluation of patients suspected of having vWD: Bleeding time Platelet count APTT FVIII:c vWF Activity CBA Blood Group RIPA (Note: Fibrinogen, FVIII:c and vWF are acute phase (Note: reactant proteins. Measurement of fibrinogen is helpful in assessing likelihood that a pt. is in an acute phase reaction at the time of testing) acute Multimer analysis Performed when Type 2vWD is suspected. A plasma sample is electrophoresed on a gel plasma to separate the multimers by size. to Type 2A:distribution of HMW vWF Type multimers lacking. multimers Type 2M: normal distribution. Discriminating tests & binding assays in vWD Discriminating Discriminating Tests Tests Plasma vWF binding Plasma assays assays Type RIPA Multimers Plt. GP1b Collagen FVIII 1 N-↓ N - N-↓ - 2A ↓ Lack Lack MMW& MMW& HMW ↓ ↓ - 2B ↑↑ Lack HMW ↑↑ ↓ - 2M ↓ N ↓ - - 2N N N - - Low 3 absent undetectable - undetectable - Von Willebrand disease (vWD) Management: The therapeutic strategies depend on accurate The diagnosis & subtyping of VWD. diagnosis Type 1: a clinical trial with DDAVP is clinical recommended. recommended. Type 3: DDAVP is unresponsive, treatment with DDAVP exogenous vWF is the choice. exogenous Type 2: DDAVP not always effective; in Type 2B, DDAVP DDAVP may worsen the thrombocytopenia & also cause spontaneous plt. aggregation. Thus, treatment with vWF concentrates is required. treatment Types of Control Plasmas used: i) ii) ii) iii) iv) Cryocheck Pooled Normal Plasma: consists of a pool of citrated human plasmas, buffered with 0.01 HEPES buffer, aliquoted and rapidly frozen. Used for determining PT/APTT only, more economic compared to IL Normal Control. RF 45: In house preparation, phospholipid free, Used for KCT and LA testing. Used Nijmegan Pool: In house preparation. Used for Bethesda assays. Pathological Trol: Abnormal control (commercial). Reporting patient test results Results of Coagulation Tests other than workups: Results are acceptable if: i) The QC samples pass ii) Raw clotting time duplicates on ‘PU’ samples Raw agree within 10% agree iii) No error codes appear next to results If results are abnormal or deviate significantly from If previous results: previous i) Discuss with senior MLSO, as further testing Discuss may be indicated may ii) Enter results with a comment if appropriate iii) Order extra tests if required iv) If abnormality is d/t a laboratory artefact, ask for If Reporting patient test results Bleeding & Thrombotic Workup Results: Results are acceptable if: i) Duplicates agree within 10%. ii) The QC samples pass. iii) If all results are correct, the MLSO must verify If them by entering initial. them iv) After verification, a senior MLSO must double After check all printed results & initial them before they are send to the results meeting. they v) All request forms & printed report are presented All to HC consultants at results meeting, where handwritten comments may be added & signed before they are despatched. before Points to ponder from observation at HC: a) General Sample collection: Use of butterfly needles and Sample coagulation syringe containers. coagulation Refrigerated centrifuge, benchtop fridges are situated Refrigerated next to coagulation analyzers for better care of reagents and samples. and Deep freezers (-45C) are available in every laboratory Deep for instant storage of samples and controls. They are periodically defrosted. periodically Small waterbath (37 C) are on almost every bench to Small thaw frozen plasma samples before analysis. Eppendorf pipetes are regularly calibrated by MLAs. MLAs are responsible for checking of temperatures and MLAs maintenance of all fridges freezers, and waterbaths. MLAs are also responsible to fill up all pipet tips, MLAs containers, tubes, etc. in every sections. containers, Points to ponder from observation at HC: 8. Lab. Coat with correct specification, i.e.the 8. sleeves fit nicely to the wrist, not dangling like ours & the buttons can be easily removed during emergencies. emergencies. 9. Biological wastes are discarded in transparent 9. plastic bags, non-biological waste in yellow plastic bags, sharps and pipet tips in used distilled water plastic containers with caps. plastic 10. Two passages of drainage from sinks, one for 10. biological/chemical wastes, the other for normal drainage. drainage. Points to ponder from observation at HC: b) Human resources: i) High degree of cooperation among staffs at all levels, High take critisms positively & professionally with view of providing better service. providing ii) Everyone knows their work, need not be told & does Everyone them responsibly. them iii) They take very short tea breaks ~15’, do not go in big They group. group. c) Results discussion & CME: c) i) Weekly results discussion session between MLSOs & Weekly consultants before results are despatched. consultants ii) Weekly departmental meeting on patients’ progress are Weekly held. held. iii) Weekly CME lectures are held. ACKNOWLEDGEMENT Sincere thanks to: 1. 2. 3. 4. 5. 6. 7. Assoc. Prof. Normah Jamaludin Dr Ramli Saad Asssoc. Prof. Zabidi Hussin Angus McCraw Anne Riddell Prof. Christine Lee Sarah Brooks, Pura Lawler and all staff of HC, Sarah Royal Free Hospital. Royal Sincere Thanks to: Last but not least, thanks to all the MLTs Last who covered my work back home during the course, especially to Syima, Maseta, Kak Sal, Wan Soriany, Miskun, Aedelley, Zulkiflee, Rohani & others. Zulkiflee, The Millenium Bridge Ancient Bath Town London Bridge Gardens of Versailles Arc de Triomphe The Stonehenge ...
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