Transcript Multi-Helath Systems

Instrumentation
Laboratory
Lab Screening for Hemorragic
Congenital Coagulopathies
Strategy and approach
Instrumentation
Laboratory
 VWD
Panel: VWF Antigen & Activity
Assays
 FII, FV, F VII, FX Clotting Assays
 FVIII, FIX, FXI, FXII Clotting Assays
 ACL TOP and ACL Elite Factor
Parallelism
 FXIII Antigen
Instrumentation
Laboratory
VWD Panel: VWF Antigen & Activity
Assays
Instrumentation
Laboratory
VWD types
Instrumentation
Laboratory
VWD Diagnosis
VWD typing
is a complex
and multitechnology
process.
Instrumentation
Laboratory
HemosIL VWD Panel
HemosIL VWF Antigen
 HemosIL VWF Activity

– Automated Latex immunoturbidrimetic
– Applications on ACL Elite/Elite Pro, ACL Advance, ACL
TOP
– VWF Antigen and Activity values assigned on the HemosIL
Plasma Calibrator and Controls
Instrumentation
Laboratory
HemosIL VWF Antigen

Latex particles coated with
rabbit polyclonal antibody
specific for VWF antigen
HemosIL VWD Panel
HemosIL VWF Activity

Latex particles coated with
anti-VWF mouse
monoclonal antibody
directed against the platelet
binding site of VWF
(Glycoprotein Ib receptor)
Plasma VWF triggers the agglutination of the latex particles
measured at 405 nm.
Instrumentation
Laboratory
HemosIL VWD panel
VWF Activity: definition
Instrumentation
Laboratory

The activity of VWF is:
 Binding
of circulating FVIII with the function
of stabilizing it and protecting from
degradation
 Adhesion
of platelets to injured vessels
– By interacting with the collagen present in the subendothelium
– This bound induces VWF to interact with platelet
receptors: Glycoprotein Ib of inactivated platelets and
glycoprotein IIb-IIIa of activated platelets
Multimer size is important for normal biological
function: the larger is the most active
Instrumentation
Laboratory

HemosIL VWF Activity
VWF: Rco
 HemosIL
VWF: Ac
– Platelet aggregation
– Latex immunoturbidimetric
– VWF causes agglutination of
stabilized platelets in the
presence of ristocetin
– Anti-VWF antibody coated on
Latex particles and directed
against the platelet binding site
of VWF (glycoprotein Ib
receptor)
– Ristocetin, in vitro, mimics the
action of collagen in modifying
the VWF conformation, thus
facilitating the binding with the
platelet receptor glycoprotein
Ib.
– The agglutination degree can
be measured in Aggregometers
(Biodata, Helena, Dade) or
Coagulation analyzers (Dade)
– Concept usually applied to
ELISA (by Axis-Shield;
Corgenix).
Instrumentation
Laboratory
Correlation with RCo
Instrumentation
Laboratory
VWD Diagnosis
Instrumentation
Laboratory
Instrumentation
Laboratory
VWF Activity: References

New Automated von Willebrand Factor Activity Assay to Distinguish
Type 1 and Type 2 von Willebrand Disease.
M. Piñol1, M. Costa, M. Sales, M.T. Canciani, A.B. Federici
XIX ISTH, Birmingham, UK July 12 – 18, 2003

Comparison of a new rapid automated VWF:Activity assay with a
visual agglutination VWF:RCo assay in screening for VWD.
JM Smith, A Bowyer, J Storr, M Makris, S Kitchen
Haemophilia 2004 : 10; 19

Comparison of a von Willebrand Factor Collagen Binding Assay with a
Latex-Enhanced Turbidimetric Immunoassay for the Determination of von
Willebrand Factor activity in Plasma.
Telting d. et al.
XVIIth International Symposium on Technologycal Innovation in
Laboratory Hemtalogy, May 13-15, 2004 Spain.
Instrumentation
Laboratory
VWF Activity: References

Determination of von Willebrand Factor Activity: Evaluation of the
HemosIL Assay in Comparison with established Procedures .
Sucker C. et al.
Clinical and Applied Thrombosis/Hemostasis, 12:3, 305 – 310, 2006

Comparison of a new Automated von willebrand Factor Activity
Assay With an Aggregation von Willebrand Ristocetin Cofactor
Activity Assay for the Diagnosis of von Willebrand Disease.
Vleeschawer etal.
Blood Coagulation & Fibrinolysis 17(5): 353-358, 2006

Evaluation of a new turbidimetric assay for von Willebrand factor
activity useful in the general screening of von Willebrand disease
M. PIÑOL, M. SALES, M. COSTA, J. SERRA, A. TOSETTO, M. T.
CANCIANI, A.B. FEDERICI
Haematologica (submitted 2006)
Instrumentation
Laboratory
Intrinsic and Extrinsic clotting Assays
Instrumentation
Laboratory
HemosIL Factor Def Plasma
Panel
Instrumentation
Laboratory
 Main
–
–
–
–
–
HemosIL Factor Def Plasma
Panel
Features
Immuno-depleted Factor Deficient Plasma
Factor level < 1% (all remaining at optimal level)
Format: 5 x 1 mL (lyo)
Stability at 2-8 °C : 24h after reconstitution
Stability on-board : 24h after reconstitution
Instrumentation
Laboratory
ACL TOP
Factor Parallelism
Instrumentation
Laboratory
 What
Factor Parallelism
is Factor Parallelism?
– Measurement of Factor Activity Levels at different sample
dilutions
– Comparison of Plasma sample results at different dilutions
with Calibration results at different dilutions


Determination of Factor Activity levels
Uncover presence of inhibitors
Instrumentation
Laboratory
 Why
Factor Parallelism
is Factor Parallelism used?
– Increase the quality of the results for factor assays
– Uncover possible interferences that might affect the test
results of the “single dilution test”
Lupus Anticoagulants
 Heparin
 Factor Inhibitors

Instrumentation
Laboratory

Factor Parallelism
How to use Factor Parallelism:
–
2-3 dilutions of the test plasma
– The result of a sample dilution should fall within the
working range of the assay
– Results of the various test plasma dilutions should
demonstrate parallelism with respect to those of the
calibration plasma
– Ideally test plasma and calibration plasma dilutions should
be the same
Instrumentation
Laboratory

Factor Parallelism
Criteria to define factor parallelism:
– No official guidelines
– Based on mathematical or statistical analysis
– Examples:
 Comparison of slopes (test plasma dilution curve vs calibration
plasma dilution curve)


Check of R2 of test plasma dilution curve
Check variance of recalculated results between first dilution
(100%) and subsequent dilutions
Instrumentation
Laboratory
Factor Parallelism
 Effects
of Heparin & Lupus
Anticoagulant interference:
– Prolongation of aPTT clotting times may result on falsely
detected low factor concentration results
– With test plasma at increasing dilutions may decrease final
heparin/Lupus concentration, thus recalculated results may
increase with the sample dilution
Instrumentation
Laboratory
 Factor
Factor Parallelism
inhibitors interference:
– Factor inhibitors may be weak or strong inhibitors,
therefore the effect of sample dilution may or may not
uncover their presence
Instrumentation
Laboratory
Factor Parallelism: ACL TOP
User definable report: may define or select up to 4 Reporting Units
Instrumentation
Laboratory

Factor Parallelism: ACL TOP
Factor Parallelism Criteria:
Instrumentation
Laboratory
ACL TOP: Normal sample
Instrumentation
Laboratory
ACL TOP: Sample with inhibitors
Instrumentation
Laboratory
ACL Elite/Elite Pro
Factor Parallelism
ACL Elite/Elite Pro:
Factor Parallelism
Instrumentation
Laboratory





3 Sample Dilutions (100%; 50%; 25%)
Dilutions Automatically done by the instruments
Calculations and Results Evaluation automatically
performed by the system and reported
1 Replicate/Level
Available for FVIII and FIX
– APTT -SP
– APTT Synthasil

Library 1.01 – 1.09
Library in development
Representation of results numerical
Instrumentation
Laboratory
Parallelism - Calculation Setup
Elite Series performs 3
Fixed Dilutions on
Samples
(1 replicate/Level)
Dilution 1 = 1:5
Dilution 2 = 1:10
Dilution 3 = 1:20
Instrumentation
Laboratory
Parallelism - Calculation Setup
User can select to display and print 4 of the
following:
CR% = Corrected Result %
 AveCR% = Average of the Corrected Results in %
 CV-CR% = CV of the Corrected Results in %
 Slope = Slope of the linear regression
(seconds vs. uncorrected % result)
 Intercept = Intercept - linear regression line
 R2 = Correlation coefficient - linear regression line

Instrumentation
Laboratory
Results for 3 dilutions are
reported in
•Seconds
•%
•Recalculated %
Parallelism Screen Report
•Mean Recalculated %
•CV Recalculated %
•Slope
•Intercept
•R2
Instrumentation
Laboratory
Parallelism Printed Detail Report
This report
displays the
individual
dilution results
plus the corrected
% activity results.
Instrumentation
Laboratory
Parallelism Printed Sample Report
This report displays
recalculated results
Instrumentation
Laboratory
FXIII Antigen Assay
Instrumentation
Laboratory
FXIII Biochemistry
Instrumentation
Laboratory
FXIII Biochemistry
Instrumentation
Laboratory
FXIII in the Coag Cascade
Instrumentation
Laboratory

FXIII Deficiency
Inherited
– Frequency 1: 5,000,000
– Types: Subunit A or Subunit B
– In type A usually undetectable levels of FXIII
– No cases of normal Factor XIII antigenic levels with impaired FXIII
activity levels have been reported so far
– The bleeding tendency typically becomes obvious when FXIII
levels are <1-2%.
– However severe bleeding episodes may occur with FXIII levels in
the range of 30% - 50%.
Instrumentation
Laboratory
FXIII Deficiency
 Acquired
–
–
–
–
–
malignant hematological diseases
severe liver disease
DIC
acute stages of inflammatory gastrointestinal disease
Henoch Schoenlein purpura
Instrumentation
Laboratory
Measurement methods
COMPETITIVE ASSAYS


Katona (Thromb Haemost 2000: 83: 268-73)
Lim W et al ( J Thromb Haemost 2004; 2: 1017-1018)
– the clot solubility tests only detect severe FXIII deficiencies with
FXIII activity levels of 0 – 3%, thus making these tests unsuitable
for detecting FXIII activity levels >5%.
– Concerns regarding the Berichrom FXIII assay analytical
sensitivity. Katona reports that the Berichrom assay is relatively
insensitive and below FXIII levels of 5% the accuracy and the
imprecision are not reliable.

As presented by the survey conducted by the FXIII working
party of European Thrombosis Research Organization (ETRO),
following a study on 72 patients a FXIII activity >5% can also be
severe (Seitz R et al. Sem thromb Hemost 1996; 22: 415-8).
Instrumentation
Laboratory
HemosIL FXIII Antigen
CONCEPT: Immunoturbidimetric assay
which measures specifically the subunit A of
the FXIII tetramer

The inherited FXIII deficiencies reported so far have decreased
antigenic and activity levels. There have been no known cases in
which the deficiency was caused by decreased activity, with
normal antigenic levels.

In addition, the very rare deficiencies affecting the subunit B
also influence the plasma levels of the subunit A (Katona E. et
al. Thromb Haemost 2000: 83: 268-73). In these cases, the
tetramer is not detectable, while the subunit A is detectable but
with low concentrations.
Instrumentation
Laboratory
HemosIL FXIII Antigen
CONCEPT: Immunoturbidimetric assay
which measures specifically the subunit A of
the FXIII tetramer

In acquired deficiencies, the measured antigenic levels of FXIIIA would be proportional to the activity levels even though they
may not be identical (Lim W et al J Thromb Haemost 2004; 2:
1017-1018) particularly after replacement therapy.
Instrumentation
Laboratory
HemosIL FXIII Antigen
Instrumentation
Laboratory
HemosIL FXIII Antigen
Instrumentation
Laboratory

HemosIL FXIII Antigen
CONCLUSIONS
– HemosIL FXIII Antigen is a liquid ready to use
immunoturbidimetric assay
– Automated on ACL Futura/Advance and ACL TOP
– Detects the Active subunit of FXIII
– Simplify and facilitates the laboratory investigation of FXIII
deficiency