Beruflich Dokumente
Kultur Dokumente
OF PLATELET MAKERS
PRE_FINAL
IMMUNOASSAY FOR THE ANTI-PLATELET FACTOR 4
(HEPARIN-INDUCED THROMBOCYTOPENIA) ANTIBODY
Plasminogen + Streptokinase
Plasmin: Streptokinase
R-pNA Plasmin: Streptokinase R-COOH + pNA
(color)
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
Clinical Significance of Plasminogen
Decreased in Increased
Thrombolytic therapy Inflammation
Thrombosis
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
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
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
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
Levels of coagulation automation
Level Description examples
Automated All reagents are ACL TOP
automatically pipetted by STA-R Evolution
the instrument. STA Compact and Compact
Specimens may or may not 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
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
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
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
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
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
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
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
Platelet Function Analyzers
PFA-100
Rapid, automated and sensitive to quantitative and
qualitative platelet abnormalities
Detects vWD and aspirin therapy