QUANTITATIVE

MEAUSREMENT OF
PLATELET MAKERS
PRE_FINAL

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 IMMUNOASSAY FOR THE ANTI-PLATELET
FACTOR 4 (HEPARIN-INDUCED
THROMBOCYTOPENIA) ANTIBODY
• Patient plasma is incubated in microtiter plate wells

that are coated with a soli-phase complex of

purified PF4 and polysulfonate, aplastic molecule
integral to plate construction that resemble heparin
• Heparin dependent anti PF4 antibodies bind the

PF4 polysulfonate complex

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 IMMUNOASSAY FOR THE ANTI-PLATELET
FACTOR 4 (HEPARIN-INDUCED
THROMBOCYTOPENIA) ANTIBODY
• Bound antibodies are detected using enzyme-
conjugated anti-human immunoglobulin G (IgG),
IgA, and IgM antibodies and a substrate
chromophore.
• Detects Ab early in the development of HIT
• Other immunoassay kits uses PF4-heparin

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 Assay for platelet activation
markers
• Elevated levels of platelet-specific proteins ß-
thromboglobulin and PF4 may accompany
thrombotic stroke or coronary thrombosis
• Diagnostica Stago, Inc (Parsipany, NJ)- Brand names
Asserachrom PF4 and Asserachrom ß-TG-produces
enzyme immunoassay kits for PF4 and ß-
thromboglobulin

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 ASSAYS
• IMMUNOASSAYS of urine 11-
dehydrothromboxane B2 are employed to
characterize in vivo platelet activation.
• Can be performed randomly
• Used to monitor aspirin therapy and to identify cases of
therapy failure or aspirin resistance

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 BETHESDA TITER FOR ANTI-
FACTOR VIII INHIBITOR
• Used to confirm the presence of and quantify an
anti factor VIII inhibitor
• Which is an IgG4-class immunoglobulin
• Uses 200uL of patient PPP
• Control specimen; 200uL of imidazole buffer at pH
7.4 mixed with 200uL of rgt normal plasma
• One bethesda unit of activity is the amount of
Activity in the mixture

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 FIBRINOLYSIS TESTS
• FDPS ( Fibrin degradation products)
• D-dimer
• Plasminogen
• TPA
• PAI-1

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 Physiology of FDPs and D-dimers
• -During coagulation, fibrin polymers become cross-
linked by factor XIIIa and binds plasma
plasminogen and tissue plasminogen activator
• Bound TPA activates plasminogen to plasmin
• Bound plasmin cleaves fibrin and yields FDPs D,
E, X and Y and D-dimer
• FDPs and D-dimer normal conc- less than 2ng/mL

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 Physiology of FDPs and D-dimers
• Fibrinolysis yields FDPs and D-dimer at conc
greater than 200ng/mL
• Increased FDPs and D-dimer-acute and chronic
DIC, systemic fibrinolysis, deep vein thrombosis
and pulmonary embolism
• FDPs and D-dimer-detected in plasma after
thrombolytic therapy

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 Principle of the Quantitative D-
Dimer Assay
• Microlatex particles in buffered saline are coated
with monoclonal anti D-dimer antibodies
• Coated particles are agglutinated by patient plasma
D-dimer
• Resultant turbidity is measured using turbidometric
or nephelometric technology
• Most mtds detect conc as low as 10ng/mL

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 Clinical significance of Quantitative
D-dimer
• D-dimer assay is essential for ruling out venous
thromboembolic disease
• Acute myocardial infarction
• Ischemic stroke
• Monitor the efficacy of Coumadin therapy
• Normal values- 250ng/mL to 500 ng/mL
• DIC, D-dimer levels- 10,000-20,000 ng/mL

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 Qualitative
D-dimer Assay
• SimpliRED D-dimer assay is a manual method that
uses visible latex particles coated with monoclonal
antibody
• Suited to low-volume or near-patient (point of care)
applications
• Clin sensitivity for pulmonary embolus (94%)

and specificity (67%)

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 Principle of Plasminogen
Chromogenic Substrate assay
• Chromogenic substrate employ synthetic
oligopeptides whose amino acid sequences are
designed to be specific for their chosen enzymes
• Plasmin hydrolyzes a bond in the oligopeptide
sequence valine-leucine-lysine
• Fluorophore or a chromophore such as para-
nitoaniline is covalently bound to carboxyl
terminus of the oligopeptide and maybe released on
digestion

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 Principle of Plasminogen
Chromogenic Substrate assay
• Streptokinase is a plasminogen activator derived
from cultures of ß-hemolytic streptococci
• Streptokinase is added to PPP where it binds and
activates plasminogen
• Streptokinase-plasmin complex reacts with
chromogenic substrate such as S-2251 to release
color whose intensity is proportional to the
plasminogen concentration.

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 Chromogenic Substrate Method for
Plasma Plasminogen
Plasminogen + Streptokinase
Plasmin: Streptokinase
R-pNA Plasmin: Streptokinase R-COOH + pNA
(color)

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 Chromogenic Substrate Method for
Plasma Plasminogen
• Reagent streptokinase activates plsaminogen to
form plasmin
• R-pNA designates a chromogenic substrate, where
R indicates one of the several choices of peptide
sequence
• pNA (para nitro aniline) is the chromophore
• In the case of plasminogen, R reperesents peptide
sequence valine-leucine-lysine
• Plasmin recognizes the Val-Leu-Lys amide
sequence as its enzymatic cleavage site, releasing
pNA, which generates yellow color

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 Clinical Significance of
Plasminogen
Plasminogen reference interval 5-
• Decreased in
13.5mg/dL Increased
• Thrombolytic therapy • Inflammation
• DIC • During pregnancy
• Hepatitis • Hemorrhage
• Cancer • systemic fibrinolysis
• Thrombosis

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 Tissue Plasminogen Activator
(TPA)
• 2 physiologic human plasminogen activators : TPA
and Urokinase
• Are serine protease that form ternnary complexes
w/bound plasminogen at the fibrin surface
activating the plasminogen and initiating thrombus
degradation
• TPA –synthesized by vascular endothelial cells;half life
in the circulation -3 minutes and its plasma
concentration averages 5ng/mL
• Urokinase- produced in the kidney and vascular
endothelial cells; half life -7 minutes and con is 2-
4ng/mL

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 Clinical Significance of Tissue
Plasminogen Activator
• Reference upper limit for TPA – 1.1 units/mL
• Upper limit for TPA antigen is 14 ng/mL
• TPA is mediator of fibrinolysis
• Decreased TPA levels- Myocardial Infarction,
stroke, deep vein thrombosis
• TPA deficiency or PAI-1 (plasminogen activator
inhibitor-1) excess is associated with deep vein
thrombosis and MI

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 Levels of coagulation automation
Level Description examples
Manual All reagents & specimens are transferred Tilt tubes
manually by the operator wireloop
Temperature is maintained by water bath
or heat block;external measuremant of the
operator is required
End point is determined visually by the
operator

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 Levels of coagulation automation
Level Description examples
Semiautomated All reagents and specimens are transferred Fibrometer
manually by the operator. Start 4
Instrument usually contains a device for Cascade M and M-4
maintaining constant 37oC temperature BFT-II
Analyzer may internally monitor the KC1 and KCA
temperature.
Instrument has mechanism to initiate
timing device automatically on addition of
final reagent and mechanism for detecting
clot formation and stopping the timer

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 Levels of coagulation automation
Level Description examples
Automated All reagents are ACL TOP
automatically pipetted by STA-R Evolution
the instrument. STA Compact and
Specimens may or may not Compact CT
be automatically pipetted. Sysmex CA-530, CA-560,
Analyzers contain CA-620, CA-660, CA-
monitoring devices and 1500,CA-7000
internal mechanism to BCS XP
maintain and monitor Coal AB
constant 37oC temperature
throughout testing
sequence
Timers are initiated and
clot formation is detected
automatically

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 Mechanical end-point detection
• Electromechanical clot detection system measure a
change in conductivity between two metal
electrodes in plasma
• Fibrometer-probe has stationary and moving electrode
• Magnetic sensor-monitors the movement of a steel ball
within the test plasma
• Principles: electromagnetic field detects oscillation of still
ball w/in plasma reagent solution
• Steel ball is positioned in inclined well, its position is
detected by magnetic sensor. As the well rotates, the ball
remains inclined position.When fibrin forms, the ball is
swept out of position. As it moves away from the sensor,
there is a break from the circuit

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 Viscosimetric
(electromechanical)clot detection
• A steel ball oscillates in an arc from one side of the
cuvette to the other
• Movement is monitored continuously with
magnetic field
• As the sample clots, viscosity rises and movement
of the steel ball is impeded
• Variation in the amplitude stops the timer and the
interval is the clotting

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 Photo optical End point Detection
(turbidometric)
• Photo-optical coagulometers detect a change in
plasma optical density (OD, light transmittance)
during clotting
• Polychromatic light is focused by a collimator and
filtered to transmit a selected wavelenght.
• Monchromatic light is transmitted by filter optics
and focused on the reaction cuvette
• As fibrin forms, opacity increases and the intensity
of light reaching the sensor decreases

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 Nephelometric end-point detection
• Modification of photo-optical end point
• Measures 90 degree or forward-angle light scatter
• Light found below passes through the sample in a
cuvette to the detector located above. As fibrin,
polymerizes, light is deflected and is detected at an
angle from the optical path

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 Chromogenic end point detection
• Employs a synthetic oligopeptide substrate
conjugated to a chromophore, para-nitoaniline
• Measures specific coagulation factor because it
exploits the factor’s enzymatic properties
• Direct chromogenic measurement-
• OD is proportional to the activity of the substance being
measured
• ex. Protein C activity is measured by a synthetic
chromogenic substrate specific for protein C

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 • Indirect chromogenic measurement-
• the CHON or analyte being measured inhibits a target
enzyme.
• It is the target enzyme that has the activity directed toward the
synthetic chromogenic substance.
• The change in the OD is inversely proportional to the
concentration of the activity of the substrate being measured-
• ex. Heparin in the anti-factor Xa assay

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 IMMUNOLOGIC LIGHT
ABSORBANCE END_POINT
• Based on Ag-Ab reactions
• Latex microparticles are coated with Abs directed
against the selected analyte (antigen)
• The increase in the light absorbance is proportional
to the antigen level

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 Platelet Function Analyzers
• PFA-100
• Rapid, automated and sensitive to quantitative and
qualitative platelet abnormalities
• Detects vWD and aspirin therapy
• Test cartridges contain membranes coated with
collagen/epinephrine or collagen/ADP to stimulate
platelet aggregation

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 Molecular Coagulation Testing
• Used for patients with thrombophilia
• Gene mutations of factor V (FV Leiden) and
prothrombin (prothrombin G20210A)
• Test Methylene-tetrahydrofolate reductase
• PCR-accurate detection of both point mutations and
single nucleotide polymorphism

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