3 Organisational Set-up Director: Professor Christine A LeeConsultant: Dr Simon BrownSenior Lecturer: Dr David PerryService Manager: Angus McCraw
4 1st. Week:28/10/02 – 1/11/02 (Morning) Attended the morning lectures with the MRCPath studentsTopics included:Introduction to the Coagulation Laboratory-Sarah BrooksNewer Techniques in Haemostasis-Anne RiddellProthrombotic State & Lab Investigation-Dr David PerryVWF and VWD-Dr Simon Brown
5 Laboratory Set-up 1) Routine Haemostasis Laboratory: Staffs:i) Pura Lawler: MLSO 2(III)ii) Sunila: MLSO 1(?)iii) Ruth: Locum MLSOiv) Marleve Dean: Trainee MLSOv) Anne Harvey: Laboratory Clerkvi) Jeremy: Locum MLAvii) Adam: Locum MLA
9 Sample Receptioni) Samples must arrive in the laboratory in sealed plastic bags, which have separate pockets for the sample and request form.ii) Samples are bar-coded, checked for suitability against the tests requested, and verified against the request forms.iii) Unsuitable samples and requests with discrepancies are noted and informed to the ward.iv) The particulars of each request are then keyed into the LIS, 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.v) The various work-ups requests, with the plasmas separated and aliquoted are kept in the deep-freezers in the various sections.vi) Platelet studies samples are sent straight to the platelet laboratory on every Tues. & Wednesdays.
10 Sample Receptionvii) For Bethesda assay, previous levels are recorded and monitored in a card for each patient.
11 Fume HoodLaminar hume hood where plasmas are separated.
14 Good Laboratory Practice in Coagulation Laboratory 1)Reagent reconstitution:i)IL Lyophilised silica for APTT- 9mls dH2O, shake vigorously, leave 30’ RT before use.ii)IL PT-HS Plus- pour entire diluent, mix gently, leave 30’RT before use.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 when not in use. DO NOT leave them unlided on the analyzer!ii)All frozen sera & reagents must be thawed at 37C in waterbath before analysis.iii) Do not analyze lysed, clotted, underfilled/overfilled samples.
16 The Nijmegan Modified Bethesda Assay for FVIII:C inhibitors Buffering the assay system with 0.1 M imidazole to pH 7.4 improves specificity and reliability.Glyoxaline buffer is used as the pre-incubation sample buffer in human Bethesda assays.These modifications allow better discrimination between positive & negative samples and improve reliability.
17 The Nijmegan Modified Bethesda Assay for FVIII:C inhibitors Interpretation and presentation of results-FVIII inhibitors may exhibit 2 forms of kineticsType 1-have a linear relationship with dilution-follows the first order kinetics with FVIII progressively neutralised until either it or the inhibitor is used up.-completely inactivate the FVIII & there is a linear relationship when the log of the residual FVIII activity is compared to the ab concentration
18 The Nijmegan Modified Bethesda Assay for FVIII:C inhibitors Type 2-inhibitors do not follow a linear relationship-exhibit a more complex order kinetics-often give similar amounts of residual FVIII with different dilutions of patient’s plasma.
20 Platelet Aggregation Tests Five agonists routinely used are ADP, adrenaline, collagen and arachidonic acid. These are adequate to allow the major platelet function disorders to be discriminated.If there is biphasic wave with 2uM ADP & adrenaline, need not proceed with higher concentrations.If patient’s platelets aggregate with 0.5mg/ml Ristocetin (indicating possible IIB VWD or Pseudo VWD), perform spontaneous aggregation for 15 minutes.
21 Platelet Aggregation Tests Calculation of results:-Most convenient and recommended to measure 3 mins after addition of agonist.Interpretation of results:-ADP at 2 uM, clearly defined primary & secondary waves can usually be seen, above 3uM, masked secondary phase; shape changed from a disc to spiky sphere.-Adrenaline, patterns of response usually similar with ADP, except primary wave does not reverse nor is it so intense to mask secondary wave; no shape change.
22 Platelet Aggregation Tests -With collagen, no primary wave occurs; response defined by duration of lag phase before onset of aggregation and its intensity.-With ristocetin, primary wave is a measure of the amount of VWF present in plasma; secondary wave is due to release of endogenous substances.-With arachidonic acid, aggregation is monophasic & preceded by a short lag phase.
23 Platelet Aggregation Tests Precautions prior to measuring platelet aggregation – see SOPFurther investigation of platelet function:-always repeat at least one occasion, if an abnormal aggregation pattern is observed.-in the presence of abnormal aggregation, further investigations include platelet nucleotides and beta-thromboglobulin;-quantitation of membrane glycoproteins for diagnosis of Bernard-Soulier syndrome & Thrombasthenia
24 Ristocetin Co-factor Assay Fresh washed platelets:-prepared from ~60 ml of blood to obtain ~20ml of PRP (sufficient to run standard & 15 patients).Advantages over lyophilised platelets:-cheap-wider aggregation range than lyophilised platelets-disadvantage: time consuming
25 Platelet Studies Laboratory Staffs:i) Anne Riddell: MLSO 3(III)ii) Sunila: MLSO 1(?)iii)Pura Lawler: MLSO 2(III)iv)Anne Harvey: Laboratory Clerkv)Jeremy: Locum MLAvi)Adam: Locum MLA
27 3) ELISA Laboratory: Tests done: a) Monoclonal Free Protein S Antigen a) Monoclonal Free Protein S Antigenb) Total Protein Sc) Protein C Agd) VWF Age) CBAf) Factor VIII:C Levels
28 Factor VIII:C Assay Done on IL 9000: -always include a blank, an abnormal & a normal control-assay test plasma in 3 dilutions, viz.1:5; 1:10 &1:20. If values obtained deviate by >10% of each other, repeat test.-check for parallelism, converging/diverging curves
29 ELISA Laboratory: Staffs: i) Saman Aghighi: MLSO 2(I) ii) Sarah Brooks: MLSO 3(II)iii) Anne Harvey: Laboratory Clerkiv) Jeremy: Locum MLAv) Adam: Locum MLA
30 4) Thrombophilia Studies: Tests done:(Sample volume 4x 3mls)a) Lupus Anticoagulantb) KCT Exner Testc) Protein S activityd) Protein C activitye) APCr Assayf) Factor V Leiden/ FII 3’UTR Multiplex PCR & Restriction Analysis
31 Lupus Anticoagulant (LA) An auto-antibody directed against , or cross-reacting with, specific chemical groups that are found in negatively charged phospholipids such as phosphatidyl serine and cardiolipin.They are usually immediate reacting, are detected by their effect on phospholipid-dependent coagulation tests.No one method has 100% reliability, usually > than one test is carried out.LA is transient in nature.
32 Sample Preparation (LA) Double centrifugationAliquot and store at –45CThe following tests are run prior to DRVVT/DRVVC:i)PT; ii)APTT; iii)APTT 50:50; iv)TT;v) Fib-C
33 Two Different LA kits are used: Manchester Reagents Russell’s Viper Venom (DRVVT & DRVVC)American Diagnostica Kit (DVVT & DVVC)If any one or both kits is/are positive, sample is considered LA positive and proceed to do KCT screen, if >1.2, Exner ensues.
34 Lupus Anticoagulant-Calculation of results Manchester Reagents:LA screen-The 20NP should give a value of s (mean 56.2)-A test/normal ratio of 1.16 or > indicates a positive result.LA confirm-The 20NP should give a value of s (mean 51.5)-If correction is > 65%, LA positive.
35 Lupus Anticoagulant-Calculation of results American Diagnostica Reagents:LA screen-The 20NP should give a value of s-Reference range for DVVT is sLA confirm-The 20NP should give a value of 29-37s-Reference Ratio of DVVTest/DVVConfirm is-A ratio of > 1.2 is considered LA positive.
36 EXNER KCT Perform an Exner screen if LA is positive. Exner KCT is positive if the ratio of NP:TP 8:2 to 20NP alone is 1.2 or greater.Then, proceed to further dilutions 10:0, 9:1, 5:5, 2:8 and 0:10 of 20NP to patient plasma.Classification of the Lupus inhibitor is determined by the shape of the curve by plot clotting times against dilutions.
37 LA test: Important points to note 20 Normal Pool used must be similarly treated as the test plasmaWarfarinised samples must be mixed with equal parts of normal pooled before tested for LA.(to compensate for the effect of warfarin on FX)If LA is negative but the prolonged APTT didn’t correct well, perform KCTHeparinised samples cannot be tested for LA.Patients on warfarin cannot be tested with an EXNER test.
38 Thrombophilia Studies: Why measure Free Protein S?-Recommendations of WHO/ISTH-Free Protein S assay have higher specificity & sensitivity for genetic defects causing Protein S deficiencies than total Protein S assay.-Only free PS is functionally active & able to bind with aPC, while the complexed form of PS is not.-There is a great overlap in the total Protein S levels between normal and those with genetic defect.
39 Thrombophilia Studies: Note:If Protein C activity is normal, need not do PC Ag.If free Protein S is normal, need not do total PS. (Free PS latex particle enhanced immunoassay, IL).If AT activity is normal, need not do AT Ag.
40 Thrombophilia Studies: Staffs:i) Sarah Brooks: MLSO 3(II)ii) Lesley Lanning: MLSO 2(II)iii) Saman Aghighi: MLSO 2(I)iv) Anna: Locum MLSOv) Anne Harvey: Laboratory Clerkvi) Jeremy: Locum MLAvii) Adam: Locum MLA
41 5) DNA Studies Laboratory: Tests done:a) Mutation Studies and DNA sequencing, etc.Staffs: i) Gillian Mellars: MLSO 3(I)ii) Anne Harvey: Laboratory Clerkiii) Jeremy: Locum MLAiv) Adam: Locum MLA
42 6) Method Development and Multimers Laboratory: Tests done:a) VWF Multimeric Studiesb) Factor VIII Binding Assayc) Special ELISAStaffs:i) Anne Riddell: MLSO 3(III)ii) Jeremy: Locum MLAiii) Adam: Locum MLA
43 Von Willebrand disease (vWD) The most common inherited bleeding disorder caused by quantitative or qualitative defects of VWF.Prevalence of 1-2 % in the general population.Different management strategies in the various types of vWD underlie the importance of classification.
44 Classification of vWDType 1 (partial quantitative deficiency, most common type)Type 2 (qualitative defect)Type 3 (total deficiency)Based on specific structural abnormalities, type 2 vWD is further classified into 4 subtypes (2A, 2B, 2N, 2M)
45 Patterns of FVIII results in vWD Notes: Type 1 is the usual heterozygous form of vWDType 3 is very rare and presents like severe haemophiliaType 2 forms are the known variant forms of vWD. These are uncommon.FVIII:cVWF:AgVWF:AcCBAAg:CBANormalNType 1 vWDN-Type 3 vWDAbsent-Type 2A vWD Type 2B vWDType 2M vWD Type 2N vWD
46 Initial laboratory evaluation of patients suspected of having vWD: Bleeding timePlatelet countAPTTFVIII:cvWF ActivityCBABlood GroupRIPA(Note: Fibrinogen, FVIII:c and vWF are acute phase reactant proteins. Measurement of fibrinogen is helpful in assessing likelihood that a pt. is in an acute phase reaction at the time of testing)
47 Multimer analysis Performed when Type 2vWD is suspected. A plasma sample is electrophoresed on a gel to separate the multimers by size.Type 2A:distribution of HMW vWF multimers lacking.Type 2M: normal distribution.
49 Von Willebrand disease (vWD) Management:The therapeutic strategies depend on accurate diagnosis & subtyping of VWD.Type 1: a clinical trial with DDAVP is recommended.Type 3: DDAVP is unresponsive, treatment with exogenous vWF is the choice.Type 2: DDAVP not always effective; in Type 2B, DDAVP may worsen the thrombocytopenia & also cause spontaneous plt. aggregation. Thus, treatment with vWF concentrates is required.
50 Types of Control Plasmas used: 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.Nijmegan Pool: In house preparation. Used for Bethesda assays.Pathological Trol: Abnormal control (commercial).
51 Reporting patient test results Results of Coagulation Tests other than workups:Results are acceptable if:The QC samples passRaw clotting time duplicates on ‘PU’ samples agree within 10%No error codes appear next to resultsIf results are abnormal or deviate significantly from previous results:Discuss with senior MLSO, as further testing may be indicatedEnter results with a comment if appropriateOrder extra tests if requiredIf abnormality is d/t a laboratory artefact, ask for a repeat sample
52 Reporting patient test results Bleeding & Thrombotic Workup Results:Results are acceptable if:Duplicates agree within 10%.The QC samples pass.If all results are correct, the MLSO must verify them by entering initial.After verification, a senior MLSO must double check all printed results & initial them before they are send to the results meeting.All request forms & printed report are presented to HC consultants at results meeting, where handwritten comments may be added & signed before they are despatched.
53 Points to ponder from observation at HC: a) GeneralSample collection: Use of butterfly needles and coagulation syringe containers.Refrigerated centrifuge, benchtop fridges are situated next to coagulation analyzers for better care of reagents and samples.Deep freezers (-45C) are available in every laboratory for instant storage of samples and controls. They are periodically defrosted.Small waterbath (37 C) are on almost every bench to thaw frozen plasma samples before analysis.Eppendorf pipetes are regularly calibrated by MLAs.MLAs are responsible for checking of temperatures and maintenance of all fridges freezers, and waterbaths.MLAs are also responsible to fill up all pipet tips, containers, tubes, etc. in every sections.
54 Points to ponder from observation at HC: 8. Lab. Coat with correct specification, i.e.the sleeves fit nicely to the wrist, not dangling like ours & the buttons can be easily removed during emergencies.9. Biological wastes are discarded in transparent plastic bags, non-biological waste in yellow plastic bags, sharps and pipet tips in used distilled water plastic containers with caps.10. Two passages of drainage from sinks, one for biological/chemical wastes, the other for normal drainage.
55 Points to ponder from observation at HC: b) Human resources:High degree of cooperation among staffs at all levels, take critisms positively & professionally with view of providing better service.Everyone knows their work, need not be told & does them responsibly.They take very short tea breaks ~15’, do not go in big group.c) Results discussion & CME:Weekly results discussion session between MLSOs & consultants before results are despatched.Weekly departmental meeting on patients’ progress are held.Weekly CME lectures are held.
56 ACKNOWLEDGEMENT Sincere thanks to: Assoc. Prof. Normah Jamaludin Dr Ramli SaadAsssoc. Prof. Zabidi HussinAngus McCrawAnne RiddellProf. Christine LeeSarah Brooks, Pura Lawler and all staff of HC, Royal Free Hospital.
57 Sincere Thanks to:Last but not least, thanks to all the MLTs who covered my work back home during the course, especially to Syima, Maseta, Kak Sal, Wan Soriany, Miskun, Aedelley, Zulkiflee, Rohani & others.