Unformatted text preview: HAEMOSTASIS &
28/10/2002 – 7/12/2002 Haemophilia Centre & Haemostasis
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
st Attended the morning lectures with the MRCPath
Topics included: Introduction to the Coagulation Laboratory-Sarah
Brooks Newer Techniques in Haemostasis-Anne Riddell Prothrombotic State & Lab Investigation-Dr David
Perry VWF and VWD-Dr Simon Brown Laboratory Set-up
1) Routine Haemostasis Laboratory:
i) Pura Lawler:
iv) Marleve Dean:
v) Anne Harvey:
vii) Adam: MLSO 2(III)
Locum MLA 1) Routine Haemostasis Laboratory:
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
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
vi) Platelet studies samples are sent straight to the platelet
laboratory on every Tues. & Wednesdays.
laboratory Sample Reception vii) For Bethesda assay, previous levels are
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
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
ii)All frozen sera & reagents must be thawed at 37C in
waterbath before analysis.
iii) Do not analyze lysed, clotted, underfilled/overfilled
samples. Fridge, Hood, Refrigerated
centrifuge, ACL 1000 The Nijmegan Modified Bethesda Assay for
Buffering the assay system with 0.1 M imidazole
to pH 7.4 improves specificity and reliability.
to 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. The Nijmegan Modified Bethesda Assay for
Interpretation and presentation of results
-FVIII inhibitors may exhibit 2 forms of kinetics
-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
activity The Nijmegan Modified Bethesda Assay for
-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.
with 2) Platelet Studies Laboratory:
Tests done: a)
e) Platelet aggregation tests
Platelet Nucleotides Bioluminescence Assay 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
If there is biphasic wave with 2uM ADP &
adrenaline, need not proceed with higher
If patient’s platelets aggregate with 0.5mg/ml
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
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
-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 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
-With arachidonic acid, aggregation is
monophasic & preceded by a short lag
phase. Platelet Aggregation Tests
Precautions prior to measuring platelet
aggregation – see SOP
aggregation Further 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 Ristocetin Co-factor Assay
Fresh washed platelets:
-prepared from ~60 ml of blood to obtain
~20ml of PRP (sufficient to run standard &
Advantages over lyophilised platelets:
-wider aggregation range than lyophilised
-disadvantage: time consuming
-disadvantage: Platelet Studies Laboratory
i) Anne Riddell:
vi)Adam: MLSO 3(III)
Locum MLA Madhavan, Saman, Lim, Anna 3) ELISA Laboratory:
f) Monoclonal Free Protein S Antigen
Total Protein S
Protein C Ag
Factor VIII:C Levels 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 ELISA Laboratory:
i) Saman Aghighi:
ii) Sarah Brooks:
iii) Anne Harvey:
v) Adam: MLSO 2(I)
Locum MLA 4) Thrombophilia Studies:
(Sample volume 4x 3mls)
f) Lupus Anticoagulant
KCT Exner Test
Protein S activity
Protein C activity
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
found in negatively charged phospholipids such as
phosphatidyl serine and cardiolipin.
They are usually immediate reacting, are detected
by their effect on phospholipid-dependent
No one method has 100% reliability, usually >
than one test is carried out.
LA is transient in nature. Sample Preparation (LA)
Double centrifugation Aliquot and store at –45C The following tests are run prior to
i)PT; ii)APTT; iii)APTT 50:50; iv)TT;
v) Fib-C Two Different LA kits are used:
Manchester Reagents Russell’s Viper
Venom (DRVVT & DRVVC)
ii) American Diagnostica Kit (DVVT &
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.
i) Lupus Anticoagulant-Calculation of results
-The 20NP should give a value of 45-65 s (mean
-A test/normal ratio of 1.16 or > indicates a positive
-The 20NP should give a value of 47-56 s (mean
-If correction is > 65%, LA positive.
-If Lupus Anticoagulant-Calculation of results
American Diagnostica Reagents:
-The 20NP should give a value of 35-45 s
-Reference range for DVVT is 27-46 s
-The 20NP should give a value of 29-37s
-Reference Ratio of DVVTest/DVVConfirm is 0.9-Reference
-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
8:2 to 20NP alone is 1.2 or greater.
8:2 Then, proceed to further dilutions 10:0, 9:1,
5:5, 2:8 and 0:10 of 20NP to patient plasma.
5:5, Classification of the Lupus inhibitor is
determined by the shape of the curve by
plot clotting times against dilutions. LA test: Important points to note
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)
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
EXNER 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
-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 Thrombophilia Studies:
Note: If Protein C activity is normal, need not do
PC If free Protein S is normal, need not do total
PS. (Free PS latex particle enhanced
immunoassay, If AT activity is normal, need not do AT
Ag. Thrombophilia Studies:
vii) Sarah Brooks:
Adam: MLSO 3(II)
Locum MLA 5) DNA Studies Laboratory:
a) Mutation Studies and DNA sequencing, etc.
i) Gillian Mellars:
ii) Anne Harvey:
iv) Adam: MLSO 3(I)
Locum MLA 6) Method Development and
c) VWF Multimeric Studies
Factor VIII Binding Assay
Special ELISA Staffs:
i) Anne Riddell:
iii) Adam: MLSO 3(III)
Locum Von Willebrand disease (vWD)
The most common inherited bleeding
disorder caused by quantitative or
qualitative defects of VWF.
qualitative Prevalence of 1-2 % in the general
population. Different management strategies in the
various types of vWD underlie the
importance of classification.
importance Classification of vWD
Type 1 (partial quantitative deficiency, most
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)
subtypes Patterns of FVIII results in vWD
Notes: Type 1 is the usual heterozygous form of vWD
Type 3 is very rare and presents like severe haemophilia
Type 2 forms are the known variant forms of vWD. These are uncommon.
Type 1 vWD
Type 3 vWD VWF:Ag
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
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
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
to separate the multimers by size.
to Type 2A:distribution of HMW vWF
multimers Type 2M: normal distribution. Discriminating tests & binding assays in
Tests Plasma vWF binding
assays Type RIPA Multimers Plt. GP1b Collagen FVIII 1 N-↓ N - N-↓ - 2A ↓ Lack
HMW ↓ ↓ - 2B ↑↑ Lack HMW ↑↑ ↓ - 2M ↓ N ↓ - - 2N N N - - Low 3 absent undetectable - undetectable - Von Willebrand disease (vWD)
The therapeutic strategies depend on accurate
diagnosis & subtyping of VWD.
Type 1: a clinical trial with DDAVP is
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.
treatment Types of Control Plasmas used:
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.
Nijmegan Pool: In house preparation. Used for
Pathological Trol: Abnormal control
(commercial). Reporting patient test results
Results of Coagulation Tests other than workups:
Results are acceptable if:
The QC samples pass
Raw clotting time duplicates on ‘PU’ samples
agree within 10%
iii) No error codes appear next to results
If results are abnormal or deviate significantly from
Discuss with senior MLSO, as further testing
may be indicated
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:
Duplicates agree within 10%.
The QC samples pass.
iii) If all results are correct, the MLSO must verify
them by entering initial.
iv) 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.
before Points to ponder from observation at
a) General Sample collection: Use of butterfly needles and
coagulation syringe containers.
coagulation Refrigerated centrifuge, benchtop fridges are situated
next to coagulation analyzers for better care of reagents
and Deep freezers (-45C) are available in every laboratory
for instant storage of samples and controls. They are
periodically 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.
containers, Points to ponder from observation at
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
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. 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
They take very short tea breaks ~15’, do not go in big
c) Results discussion & CME:
Weekly results discussion session between MLSOs &
consultants before results are despatched.
Weekly departmental meeting on patients’ progress are
Weekly CME lectures are held. ACKNOWLEDGEMENT
Sincere thanks to:
7. Assoc. Prof. Normah Jamaludin
Dr Ramli Saad
Asssoc. Prof. Zabidi Hussin
Prof. Christine Lee
Sarah Brooks, Pura Lawler and all staff of HC,
Royal Free Hospital.
Royal 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.
Zulkiflee, The Millenium Bridge Ancient Bath Town London Bridge Gardens of Versailles Arc de Triomphe The Stonehenge ...
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- Platelet, Von Willebrand disease, platelet aggregation tests, Locum MLA