HAEMOSTASIS & THROMBOSIS

ATTACHMENT COURSE

28/10/2002 – 7/12/2002

Haemophilia Centre & Haemostasis Haemophilia Centre & Haemostasis Unit, Royal Free HospitalUnit, Royal Free Hospital

RFH

Organisational Set-up

Director: Professor Christine A LeeDirector: Professor Christine A Lee Consultant: Dr Simon BrownConsultant: Dr Simon Brown Senior Lecturer: Dr David PerrySenior Lecturer: Dr David Perry Service Manager: Angus McCrawService Manager: Angus McCraw

1st. Week:28/10/02 – 1/11/02 (Morning)Attended the morning lectures with the MRCPath Attended the morning lectures with the MRCPath

studentsstudentsTopics included:Topics included: Introduction to the Coagulation Laboratory-Sarah Introduction to the Coagulation Laboratory-Sarah

BrooksBrooks Newer Techniques in Haemostasis-Anne RiddellNewer Techniques in Haemostasis-Anne Riddell Prothrombotic State & Lab Investigation-Dr Prothrombotic State & Lab Investigation-Dr

David PerryDavid Perry VWF and VWD-Dr Simon BrownVWF and VWD-Dr Simon Brown

Laboratory Set-up1) Routine Haemostasis Laboratory:

Staffs:Staffs:  

i)i) Pura Lawler:Pura Lawler: MLSO 2(III)MLSO 2(III)ii)ii) Sunila:Sunila: MLSO 1(?)MLSO 1(?)iii)iii) Ruth:Ruth: Locum MLSO Locum MLSOiv)iv) Marleve Dean:Marleve Dean: Trainee MLSOTrainee MLSO

v) Anne Harvey:v) Anne Harvey: Laboratory ClerkLaboratory Clerkvi)vi) Jeremy:Jeremy: Locum MLA Locum MLAvii)vii) Adam:Adam: Locum MLALocum MLA

1) Routine Haemostasis Laboratory:

Tests done:Tests done: a)    PT/APTTa)    PT/APTT b)   TT/RTb)   TT/RT c)    FIB-Cc)    FIB-C d)   D-Dimerd)   D-Dimer e)    Factors Assays: FVII, FVIII, FIX, FXIe)    Factors Assays: FVII, FVIII, FIX, FXI f)     Inhibitor Screensf)     Inhibitor Screens g)    Mixing Testsg)    Mixing Tests h) Nijmegan Inhibitor Assays h) Nijmegan Inhibitor Assays i)i) Others: Fletcher, Fitzgerald, PF4, etc.Others: Fletcher, Fitzgerald, PF4, etc.

Reception Counter

Adam, Pura, Lim, Marleve

Sample Reception i)  Samples must arrive in the laboratory in sealed plastic bags, i)  Samples must arrive in the laboratory in sealed plastic bags, which which

have separate pockets for the sample and request have separate pockets for the sample and request form.form. ii)    Samples are bar-coded, checked for suitability against the ii)    Samples are bar-coded, checked for suitability against the tests tests

requested, and verified against the request forms.requested, and verified against the request forms. iii)   Unsuitable samples and requests with discrepancies are iii)   Unsuitable samples and requests with discrepancies are noted noted

and informed to the ward.and informed to the ward. iv)    The particulars of each request are then keyed into the LIS, iv)    The particulars of each request are then keyed into the LIS, first first

time requests that are not previously tested are time requests that are not previously tested are stamped “PU” stamped “PU” (previously untested) on the request forms (previously untested) on the request forms and are required to be and are required to be assayed in duplicate.assayed in duplicate.

v)     The various work-ups requests, with the plasmas separated v)     The various work-ups requests, with the plasmas separated and and aliquoted are kept in the deep-freezers in the aliquoted are kept in the deep-freezers in the various sections.various sections.

vi)    Platelet studies samples are sent straight to the platelet vi)    Platelet studies samples are sent straight to the platelet laboratory on every Tues. & Wednesdays.laboratory on every Tues. & Wednesdays.

Sample Reception

vii) For Bethesda assay, previous levels are vii) For Bethesda assay, previous levels are recorded and monitored in a card for each recorded and monitored in a card for each patient. patient.

Fume Hood

ACL Futura Coagulation Analyzer

Bench-top fridge

Good Laboratory Practice in Coagulation Laboratory 1)Reagent reconstitution:1)Reagent reconstitution: i)IL Lyophilised silica for APTT- 9mls dH2O, shake i)IL Lyophilised silica for APTT- 9mls dH2O, shake

vigorously, leave 30’ RT before use.vigorously, leave 30’ RT before use. ii)IL PT-HS Plus- pour entire diluent, mix gently, leave ii)IL PT-HS Plus- pour entire diluent, mix gently, leave

30’RT before use.30’RT before use. iii)NP-1.0ml dH2O, leave 30’RT before use.iii)NP-1.0ml dH2O, leave 30’RT before use.

2)Proper handling of reagents, NP and samples:2)Proper handling of reagents, NP and samples: i)Keep all reconstituted reagents & sera at 4C & cover lid i)Keep all reconstituted reagents & sera at 4C & cover lid

when not in use. DO NOT leave them unlided on the 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 frozen sera & reagents must be thawed at 37C in waterbath before analysis.waterbath before analysis.

iii) Do not analyze lysed, clotted, underfilled/overfilled iii) Do not analyze lysed, clotted, underfilled/overfilled 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 the assay system with 0.1 M imidazole to pH 7.4 improves specificity and reliability.to pH 7.4 improves specificity and reliability.

Glyoxaline buffer is used as the pre-incubation Glyoxaline buffer is used as the pre-incubation sample buffer in human Bethesda assays.sample buffer in human Bethesda assays.

These modifications allow better discrimination These modifications allow better discrimination between positive & negative samples and improve between positive & negative samples and improve reliability. reliability.

The Nijmegan Modified Bethesda Assay for FVIII:C inhibitors

Interpretation and presentation of resultsInterpretation and presentation of results-FVIII inhibitors may exhibit 2 forms of kinetics-FVIII inhibitors may exhibit 2 forms of kineticsType 1 Type 1 -have a linear relationship with dilution -have a linear relationship with dilution -follows the first order kinetics with FVIII -follows the first order kinetics with FVIII

progressively neutralised until either it or the progressively neutralised until either it or the inhibitor is used up.inhibitor is used up.

-completely inactivate the FVIII & there is a linear -completely inactivate the FVIII & there is a linear relationship when the log of the residual FVIII relationship when the log of the residual FVIII activity is compared to the ab concentrationactivity is compared to the ab concentration

The Nijmegan Modified Bethesda Assay for FVIII:C inhibitorsType 2 Type 2

-inhibitors do not follow a linear relationship-inhibitors do not follow a linear relationship

-exhibit a more complex order kinetics-exhibit a more complex order kinetics

-often give similar amounts of residual FVIII -often give similar amounts of residual FVIII with different dilutions of patient’s plasma.with different dilutions of patient’s plasma.

2) Platelet Studies Laboratory:

Tests done:Tests done:

a)  Platelet aggregation testsa)  Platelet aggregation tests b)  RiCOF Assayb)  RiCOF Assay c)  PFA c)  PFA d)  TEG testd)  TEG test e)   Platelet Nucleotides Bioluminescence Assaye)   Platelet Nucleotides Bioluminescence Assay   

Platelet Aggregation Tests Five agonists routinely used are ADP, adrenaline, Five agonists routinely used are ADP, adrenaline,

collagen and arachidonic acid. These are adequate collagen and arachidonic acid. These are adequate to allow the major platelet function disorders to be to allow the major platelet function disorders to be discriminated.discriminated.

If there is biphasic wave with 2uM ADP & If there is biphasic wave with 2uM ADP & adrenaline, need not proceed with higher adrenaline, need not proceed with higher concentrations.concentrations.

If patient’s platelets aggregate with 0.5mg/ml If patient’s platelets aggregate with 0.5mg/ml Ristocetin (indicating possible IIB VWD or Ristocetin (indicating possible IIB VWD or Pseudo VWD), perform spontaneous aggregation Pseudo VWD), perform spontaneous aggregation for 15 minutes.for 15 minutes.

Platelet Aggregation TestsCalculation of results:Calculation of results:-Most convenient and recommended to measure 3 mins -Most convenient and recommended to measure 3 mins

after addition of agonist.after addition of agonist.Interpretation of results:Interpretation of results:-ADP at 2 uM, clearly defined primary & secondary -ADP at 2 uM, clearly defined primary & secondary

waves can usually be seen, above 3uM, masked waves can usually be seen, above 3uM, masked secondary phase; shape changed from a disc to spiky secondary phase; shape changed from a disc to spiky sphere.sphere.

-Adrenaline, patterns of response usually similar with -Adrenaline, patterns of response usually similar with ADP, except primary wave does not reverse nor is it so ADP, except primary wave does not reverse nor is it so intense to mask secondary wave; no shape change.intense to mask secondary wave; no shape change.

Platelet Aggregation Tests-With collagen, no primary wave occurs; -With collagen, no primary wave occurs;

response defined by duration of lag phase response defined by duration of lag phase before onset of aggregation and its intensity.before onset of aggregation and its intensity.

-With ristocetin, primary wave is a measure of -With ristocetin, primary wave is a measure of the amount of VWF present in plasma; the amount of VWF present in plasma; secondary wave is due to release of secondary wave is due to release of endogenous substances.endogenous substances.

-With arachidonic acid, aggregation is -With arachidonic acid, aggregation is monophasic & preceded by a short lag phase.monophasic & preceded by a short lag phase.

Platelet Aggregation Tests Precautions prior to measuring platelet Precautions prior to measuring platelet

aggregationaggregation – see SOP – see SOP Further investigation of platelet function:Further investigation of platelet function:

-always repeat at least one occasion, if an -always repeat at least one occasion, if an abnormal aggregation pattern is observed.abnormal aggregation pattern is observed.

-in the presence of abnormal aggregation, -in the presence of abnormal aggregation, further investigations include platelet further investigations include platelet nucleotides and beta-thromboglobulin;nucleotides and beta-thromboglobulin;

-quantitation of membrane glycoproteins -quantitation of membrane glycoproteins for diagnosis of Bernard-Soulier syndrome for diagnosis of Bernard-Soulier syndrome & Thrombasthenia& Thrombasthenia

Ristocetin Co-factor AssayFresh washed platelets:Fresh washed platelets:-prepared from ~60 ml of blood to obtain -prepared from ~60 ml of blood to obtain

~20ml of PRP (sufficient to run standard & ~20ml of PRP (sufficient to run standard & 15 patients).15 patients).

Advantages over lyophilised platelets:Advantages over lyophilised platelets:-cheap -cheap -wider aggregation range than lyophilised -wider aggregation range than lyophilised

plateletsplatelets-disadvantage: time consuming -disadvantage: time consuming

Platelet Studies Laboratory

Staffs:Staffs:

  

i)i) Anne Riddell:Anne Riddell: MLSO 3(III)MLSO 3(III)

ii)ii)Sunila:Sunila: MLSO 1(?)MLSO 1(?)

iii)Pura Lawler:iii)Pura Lawler: MLSO 2(III)MLSO 2(III)

iv)Anne Harvey:iv)Anne Harvey: Laboratory ClerkLaboratory Clerk

v)Jeremy:v)Jeremy: Locum MLALocum MLA

vi)Adam:vi)Adam: Locum MLALocum MLA

Madhavan, Saman, Lim, Anna

3) ELISA Laboratory:

Tests done:Tests done:  a)    Monoclonal Free Protein S Antigen a)    Monoclonal Free Protein S Antigen b)    Total Protein S b)    Total Protein S c)    Protein C Ag c)    Protein C Ag d)    VWF Ag d)    VWF Ag e)    CBAe)    CBAf) Factor VIII:C Levels f) Factor VIII:C Levels           

Factor VIII:C Assay

Done on IL 9000:Done on IL 9000:

-always include a blank, an abnormal & -always include a blank, an abnormal & a normal controla normal control

-assay test plasma in 3 dilutions, viz.1:5; -assay test plasma in 3 dilutions, viz.1:5; 1:10 &1:20. If values obtained deviate 1:10 &1:20. If values obtained deviate by >10% of each other, repeat test.by >10% of each other, repeat test.

-check for parallelism, -check for parallelism, converging/diverging curvesconverging/diverging curves

ELISA Laboratory:

Staffs:Staffs:

    i)i) Saman Aghighi:Saman Aghighi: MLSO 2(I) MLSO 2(I)

ii) Sarah Brooks:ii) Sarah Brooks: MLSO 3(II) MLSO 3(II)

iii) Anne Harvey:iii) Anne Harvey: Laboratory Clerk Laboratory Clerk

iv)iv) Jeremy:Jeremy: Locum MLA Locum MLA

v)v) Adam:Adam: Locum MLA Locum MLA

4) Thrombophilia Studies:Tests done:Tests done:(Sample volume 4x 3mls)(Sample volume 4x 3mls)  a)       Lupus Anticoagulanta)       Lupus Anticoagulantb)       KCT Exner Testb)       KCT Exner Testc)       Protein S activityc)       Protein S activityd)       Protein C activityd)       Protein C activitye)       APCr Assaye)       APCr Assayf)  Factor V Leiden/ FII 3’UTR Multiplex PCR f)  Factor V Leiden/ FII 3’UTR Multiplex PCR

& Restriction Analysis & Restriction Analysis

Lupus Anticoagulant (LA) An auto-antibody directed against , or cross-An auto-antibody directed against , or cross-

reacting with, specific chemical groups that are reacting with, specific chemical groups that are found in negatively charged phospholipids such as found in negatively charged phospholipids such as phosphatidyl serine and cardiolipin.phosphatidyl serine and cardiolipin.

They are usually immediate reacting, are detected They are usually immediate reacting, are detected by their effect on phospholipid-dependent by their effect on phospholipid-dependent coagulation tests.coagulation tests.

No one method has 100% reliability, usually > No one method has 100% reliability, usually > than one test is carried out.than one test is carried out.

LA is transient in nature.LA is transient in nature.

Sample Preparation (LA)

Double centrifugationDouble centrifugation Aliquot and store at –45CAliquot and store at –45C

The following tests are run prior to The following tests are run prior to DRVVT/DRVVC:DRVVT/DRVVC:

i)PT; ii)APTT; iii)APTT 50:50; iv)TT; i)PT; ii)APTT; iii)APTT 50:50; iv)TT; v) Fib-C v) Fib-C

Two Different LA kits are used:

i)i) Manchester Reagents Russell’s Viper Manchester Reagents Russell’s Viper Venom (DRVVT & DRVVC)Venom (DRVVT & DRVVC)

ii)ii) American Diagnostica Kit (DVVT & American Diagnostica Kit (DVVT & DVVC)DVVC)

If any one or both kits is/are positive, sample If any one or both kits is/are positive, sample is considered LA positive and proceed to is considered LA positive and proceed to do KCT screen, if >1.2, Exner ensues.do KCT screen, if >1.2, Exner ensues.

Lupus Anticoagulant-Calculation of results

Manchester Reagents:Manchester Reagents: LA screenLA screen -The 20NP should give a value of 45-65 s (mean 56.2)-The 20NP should give a value of 45-65 s (mean 56.2) -A test/normal ratio of 1.16 or > indicates a positive -A test/normal ratio of 1.16 or > indicates a positive

result.result.LA confirmLA confirm -The 20NP should give a value of 47-56 s (mean 51.5)-The 20NP should give a value of 47-56 s (mean 51.5) -If correction is > 65%, LA positive.-If correction is > 65%, LA positive.

Lupus Anticoagulant-Calculation of resultsAmerican Diagnostica Reagents: American Diagnostica Reagents: LA screenLA screen -The 20NP should give a value of 35-45 s -The 20NP should give a value of 35-45 s -Reference range for DVVT is 27-46 s-Reference range for DVVT is 27-46 sLA confirmLA confirm -The 20NP should give a value of 29-37s-The 20NP should give a value of 29-37s -Reference Ratio of DVVTest/DVVConfirm is 0.9--Reference Ratio of DVVTest/DVVConfirm is 0.9-

1.21.2 -A ratio of > 1.2 is considered LA positive.-A ratio of > 1.2 is considered LA positive.

EXNER KCT Perform an Exner screen if LA is positive.Perform an Exner screen if LA is positive. Exner KCT is positive if the ratio of NP:TP Exner KCT is positive if the ratio of NP:TP

8:2 to 20NP alone is 1.2 or greater.8:2 to 20NP alone is 1.2 or greater. Then, proceed to further dilutions 10:0, 9:1, Then, proceed to further dilutions 10:0, 9:1,

5:5, 2:8 and 0:10 of 20NP to patient plasma.5:5, 2:8 and 0:10 of 20NP to patient plasma. Classification of the Lupus inhibitor is Classification of the Lupus inhibitor is

determined by the shape of the curve by determined by the shape of the curve by plot clotting times against dilutions. plot clotting times against dilutions.

LA test: Important points to notei) 20 Normal Pool used must be similarly treated

as the test plasmaii) 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) If LA is negative but the prolonged APTT didn’t correct well, perform KCT

iv) Heparinised samples cannot be tested for LA.v)v) Patients on warfarin cannot be tested with an Patients on warfarin cannot be tested with an

EXNER test.EXNER test.

Thrombophilia Studies:Why measure Free Protein S?Why measure Free Protein S?-Recommendations of WHO/ISTH-Recommendations of WHO/ISTH-Free Protein S assay have higher specificity & -Free Protein S assay have higher specificity &

sensitivity for genetic defects causing Protein S sensitivity for genetic defects causing Protein S deficiencies than total Protein S assay.deficiencies than total Protein S assay.

-Only free PS is functionally active & able to -Only free PS is functionally active & able to bind with aPC, while the complexed form of PS bind with aPC, while the complexed form of PS is not.is not.

-There is a great overlap in the total Protein S -There is a great overlap in the total Protein S levels between normal and those with genetic levels between normal and those with genetic defect.defect.

Thrombophilia Studies:

Note:Note: If Protein C activity is normal, need not do If Protein C activity is normal, need not do

PC Ag.PC Ag. If free Protein S is normal, need not do total If free Protein S is normal, need not do total

PS. (Free PS latex particle enhanced PS. (Free PS latex particle enhanced immunoassay, IL).immunoassay, IL).

If AT activity is normal, need not do AT If AT activity is normal, need not do AT Ag.Ag.

Thrombophilia Studies:

Staffs:Staffs:  i)i) Sarah Brooks:Sarah Brooks: MLSO 3(II)MLSO 3(II)ii)ii) Lesley Lanning:Lesley Lanning: MLSO 2(II)MLSO 2(II)iii)iii) Saman Aghighi:Saman Aghighi: MLSO 2(I)MLSO 2(I)iv)iv) Anna:Anna: Locum MLSOLocum MLSOv)v) Anne Harvey:Anne Harvey: Laboratory ClerkLaboratory Clerkvi)vi) Jeremy:Jeremy: Locum MLALocum MLAvii)vii) Adam:Adam: Locum MLALocum MLA

5) DNA Studies Laboratory:

Tests done:Tests done:  

a) Mutation Studies and DNA sequencing, etc.a) Mutation Studies and DNA sequencing, etc.  Staffs:Staffs:    i)i) Gillian Mellars:Gillian Mellars: MLSO 3(I)MLSO 3(I) ii)ii) Anne Harvey:Anne Harvey: Laboratory ClerkLaboratory Clerkiii)iii) Jeremy:Jeremy: Locum MLALocum MLAiv)iv) Adam:Adam: Locum MLALocum MLA

6) Method Development and Multimers Laboratory:

Tests done:Tests done:  a)        VWF Multimeric Studiesa)        VWF Multimeric Studiesb)        Factor VIII Binding Assayb)        Factor VIII Binding Assayc)         Special ELISAc)         Special ELISA  Staffs:Staffs:  i)i) Anne Riddell:Anne Riddell: MLSO 3(III)MLSO 3(III)

ii)ii) Jeremy:Jeremy: Locum MLALocum MLAiii)iii) Adam:Adam: Locum MLA Locum MLA

Von Willebrand disease (vWD)

The most common inherited bleeding The most common inherited bleeding disorder caused by quantitative or disorder caused by quantitative or qualitative defects of VWF.qualitative defects of VWF.

Prevalence of 1-2 % in the general Prevalence of 1-2 % in the general population.population.

Different management strategies in the Different management strategies in the various types of vWD underlie the various types of vWD underlie the importance of classification.importance of classification.

Classification of vWD Type 1 (partial quantitative deficiency, most Type 1 (partial quantitative deficiency, most

common type)common type) Type 2 (qualitative defect)Type 2 (qualitative defect) Type 3 (total deficiency)Type 3 (total deficiency)

Based on specific structural abnormalities, Based on specific structural abnormalities, type 2 vWD is further classified into 4 type 2 vWD is further classified into 4 subtypes (2A, 2B, 2N, 2M)subtypes (2A, 2B, 2N, 2M)

Patterns of FVIII results in vWDNotes: Notes: Type 1 is the usual heterozygous form of vWD Type 1 is the usual heterozygous form of vWD

Type 3 is very rare and presents like severe haemophiliaType 3 is very rare and presents like severe haemophilia

Type 2 forms are the known variant forms of vWD. These are uncommon.Type 2 forms are the known variant forms of vWD. These are uncommon.

FVIII:cFVIII:c VWF:AgVWF:Ag VWF:AcVWF:Ac CBACBA Ag:CBA Ag:CBA

NormalNormal NN NN NN NN NN

Type 1 vWDType 1 vWD N-N- NN

Type 3 vWDType 3 vWD AbsentAbsent AbsentAbsent AbsentAbsent AbsentAbsent --

Type 2A vWDType 2A vWD N-N- N-N- N-N-

Type 2B vWDType 2B vWD N-N- N-N- N-N-

Type 2M vWDType 2M vWD N-N- N-N- N-N- NN

Type 2N vWDType 2N vWD NN NN NN NN

Initial laboratory evaluation of patients suspected of having vWD: Bleeding timeBleeding time Platelet countPlatelet count APTTAPTT FVIII:cFVIII:c vWF ActivityvWF Activity CBACBA Blood GroupBlood Group RIPARIPA(Note: Fibrinogen, FVIII:c and vWF are acute phase (Note: Fibrinogen, FVIII:c and vWF are acute phase

reactant proteins. Measurement of fibrinogen is reactant proteins. Measurement of fibrinogen is helpful in assessing likelihood that a pt. is in an helpful in assessing likelihood that a pt. is in an acute phase reaction at the time of testing)acute phase reaction at the time of testing)

Multimer analysis

Performed when Type 2vWD is suspected.Performed when Type 2vWD is suspected. A plasma sample is electrophoresed on a A plasma sample is electrophoresed on a

gel to separate the multimers by size.gel to separate the multimers by size. Type 2A:distribution of HMW vWF Type 2A:distribution of HMW vWF

multimers lacking.multimers lacking. Type 2M: normal distribution.Type 2M: normal distribution.

Discriminating tests & binding assays in vWD

Discriminating Discriminating TestsTests

Plasma vWF binding Plasma vWF binding assaysassays

TypeType RIPARIPA MultimersMultimers Plt. GP1bPlt. GP1b CollagenCollagen FVIIIFVIII

11 N-N-↓↓ NN -- N-↓N-↓ --

2A2A ↓↓ Lack Lack MMW&MMW&

HMWHMW

↓↓ ↓↓ --

2B2B ↑ ↑↑ ↑ Lack HMWLack HMW ↑ ↑↑ ↑ ↓↓ --

2M2M ↓↓ NN ↓↓ -- --

2N2N NN NN -- -- LowLow

33 absentabsent undetectableundetectable -- undetectableundetectable --

Von Willebrand disease (vWD)

Management:Management:The therapeutic strategies depend on accurate The therapeutic strategies depend on accurate

diagnosis & subtyping of VWD.diagnosis & subtyping of VWD.

Type 1Type 1: a clinical trial with DDAVP is : a clinical trial with DDAVP is recommended.recommended.

Type 3Type 3: DDAVP is unresponsive, treatment with : DDAVP is unresponsive, treatment with exogenous vWF is the choice.exogenous vWF is the choice.

Type 2Type 2: DDAVP not always effective; in Type 2B, : DDAVP not always effective; in Type 2B, DDAVP may worsen the thrombocytopenia & DDAVP may worsen the thrombocytopenia & also cause spontaneous plt. aggregation. Thus, also cause spontaneous plt. aggregation. Thus, treatment with vWF concentrates is required.treatment with vWF concentrates is required.

Types of Control Plasmas used:i)i) Cryocheck Pooled Normal Plasma:Cryocheck Pooled Normal Plasma: consists of a consists of a

pool of citrated human plasmas, buffered with 0.01 pool of citrated human plasmas, buffered with 0.01 HEPES buffer, aliquoted and rapidly frozen. Used HEPES buffer, aliquoted and rapidly frozen. Used for determining PT/APTT only, more economic for determining PT/APTT only, more economic compared to IL Normal Control. compared to IL Normal Control.

ii)ii) RF 45:RF 45: In house preparation, phospholipid free, In house preparation, phospholipid free, Used for KCT and LA testing.Used for KCT and LA testing.

iii)iii) Nijmegan Pool:Nijmegan Pool: In house preparation. Used for In house preparation. Used for Bethesda assays. Bethesda assays.

iv)iv) Pathological Trol:Pathological Trol: Abnormal control Abnormal control (commercial). (commercial).

Reporting patient test resultsResults of Coagulation Tests other than workups:Results of Coagulation Tests other than workups:Results are acceptable if:Results are acceptable if:i)i) The QC samples passThe QC samples passii)ii) Raw clotting time duplicates on ‘PU’ samples agree Raw clotting time duplicates on ‘PU’ samples agree

within 10%within 10%iii)iii) No error codes appear next to resultsNo error codes appear next to resultsIf results are abnormal or deviate significantly from If results are abnormal or deviate significantly from

previous results:previous results:i)i) Discuss with senior MLSO, as further testing may Discuss with senior MLSO, as further testing may

be indicatedbe indicatedii)ii) Enter results with a comment if appropriateEnter results with a comment if appropriateiii)iii) Order extra tests if requiredOrder extra tests if requirediv)iv) If abnormality is d/t a laboratory artefact, ask for a If abnormality is d/t a laboratory artefact, ask for a

repeat samplerepeat sample

Reporting patient test resultsBleeding & Thrombotic Workup Results:Bleeding & Thrombotic Workup Results:Results are acceptable if:Results are acceptable if:i)i) Duplicates agree within 10%.Duplicates agree within 10%.ii)ii) The QC samples pass.The QC samples pass.iii)iii) If all results are correct, the MLSO must verify If all results are correct, the MLSO must verify

them by entering initial.them by entering initial.iv)iv) After verification, a senior MLSO must double After verification, a senior MLSO must double

check all printed results & initial them before check all printed results & initial them before they are send to the results meeting.they are send to the results meeting.

v)v) All request forms & printed report are presented All request forms & printed report are presented to HC consultants at results meeting, where to HC consultants at results meeting, where handwritten comments may be added & signed handwritten comments may be added & signed before they are despatched.before they are despatched.

Points to ponder from observation at HC:a) Generala) General Sample collection: Use of butterfly needles and Sample collection: Use of butterfly needles and

coagulation syringe containers.coagulation syringe containers. Refrigerated centrifuge, benchtop fridges are situated Refrigerated centrifuge, benchtop fridges are situated

next to coagulation analyzers for better care of reagents next to coagulation analyzers for better care of reagents and samples.and samples.

Deep freezers (-45C) are available in every laboratory Deep freezers (-45C) are available in every laboratory for instant storage of samples and controls. They are for instant storage of samples and controls. They are periodically defrosted.periodically defrosted.

Small waterbath (37 C) are on almost every bench to Small waterbath (37 C) are on almost every bench to thaw frozen plasma samples before analysis. thaw frozen plasma samples before analysis.

Eppendorf pipetes are regularly calibrated by MLAs.Eppendorf pipetes are regularly calibrated by MLAs. MLAs are responsible for checking of temperatures and MLAs are responsible for checking of temperatures and

maintenance of all fridges freezers, and waterbaths. maintenance of all fridges freezers, and waterbaths. MLAs are also responsible to fill up all pipet tips, MLAs are also responsible to fill up all pipet tips,

containers, tubes, etc. in every sections.containers, tubes, etc. in every sections.

Points to ponder from observation at HC:8. Lab. Coat with correct specification, i.e.the 8. Lab. Coat with correct specification, i.e.the

sleeves fit nicely to the wrist, not dangling like sleeves fit nicely to the wrist, not dangling like ours & the buttons can be easily removed during ours & the buttons can be easily removed during emergencies.emergencies.

9. Biological wastes are discarded in transparent 9. Biological wastes are discarded in transparent plastic bags, non-biological waste in yellow plastic bags, non-biological waste in yellow plastic bags, sharps and pipet tips in used distilled plastic bags, sharps and pipet tips in used distilled water plastic containers with caps.water plastic containers with caps.

10. Two passages of drainage from sinks, one for 10. Two passages of drainage from sinks, one for biological/chemical wastes, the other for normal biological/chemical wastes, the other for normal drainage.drainage.

Points to ponder from observation at HC:b) Human resources:b) Human resources:i)i) High degree of cooperation among staffs at all levels, take critisms High degree of cooperation among staffs at all levels, take critisms

positively & professionally with view of providing better service.positively & professionally with view of providing better service.ii)ii) Everyone knows their work, need not be told & does them Everyone knows their work, need not be told & does them

responsibly.responsibly.iii)iii) They take very short tea breaks ~15’, do not go in big group.They take very short tea breaks ~15’, do not go in big group.

c) Results discussion & CME:c) Results discussion & CME:i)i) Weekly results discussion session between MLSOs & consultants Weekly results discussion session between MLSOs & consultants

before results are despatched.before results are despatched.ii)ii) Weekly departmental meeting on patients’ progress are held.Weekly departmental meeting on patients’ progress are held.iii)iii) Weekly CME lectures are held.Weekly CME lectures are held.

ACKNOWLEDGEMENT

Sincere thanks to:Sincere thanks to:1.1. Assoc. Prof. Normah JamaludinAssoc. Prof. Normah Jamaludin2.2. Dr Ramli SaadDr Ramli Saad3.3. Asssoc. Prof. Zabidi HussinAsssoc. Prof. Zabidi Hussin4.4. Angus McCrawAngus McCraw5.5. Anne RiddellAnne Riddell6.6. Prof. Christine LeeProf. Christine Lee7.7. Sarah Brooks, Pura Lawler and all staff of HC, Sarah Brooks, Pura Lawler and all staff of HC,

Royal Free Hospital.Royal Free Hospital.

Sincere Thanks to:

Last but not least, thanks to all the MLTs Last but not least, thanks to all the MLTs who covered my work back home during who covered my work back home during the course, especially to Syima, Maseta, the course, especially to Syima, Maseta, Kak Sal, Wan Soriany, Miskun, Aedelley, Kak Sal, Wan Soriany, Miskun, Aedelley, Zulkiflee, Rohani & others.Zulkiflee, Rohani & others.

The Millenium Bridge

Ancient Bath Town

London Bridge

Gardens of Versailles

Arc de Triomphe

The Stonehenge