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  文件名称:  Thrombin Generation Kit Insert February HAEMOSCAN BV
  公司名称:  上海拜力生物科技有限公司
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Thrombin Generation Kit Insert February HAEMOSCAN BV

 COMPANY WITH
 QUALITY MANAGEMENT SYSTEM
 CERTIFIED BY DNV GL
 = ISO 9001 =
 Thrombin Generation
 Assay
 Kit Insert
 Version: February 2018
Summary
Thrombin is a key enzyme of the coagulation cascade. Its measurement
gives direct information about the thrombogenicity of a biomaterial (i.e.,
its ability to form blood clots). In normal plasma, thrombin is captured
into the fibrin meshwork and is rapidly inactivated by antithrombin III or
other antiproteases. The short half-life of thrombin hampers its accurate
enzymatic determination. Therefore, HaemoScan has designed the
Thrombin Generation Assay [1], which is based on a special plasma
product that enables the determination of thrombin activity in an
incubation medium after this has been exposed to a biomaterial. This
method is suited to evaluate the haemocompatibility of biomaterials and
medical devices according to the international standard ISO 10993-
4:2002.
[1] W. van Oeveren, J. Haan, P. Lagerman, and P. Schoen, “Comparison
of coagulation activity tests in vitro for selected biomaterials.,” Artificial
organs, vol. 26, no. 6, pp. 506–11, Jun. 2002.
Introduction
Interactions between blood components and biomaterials may activate
the clotting cascade. Activated factor XII, the Hageman factor (FXIIa)
initiates the intrinsic pathway. Depending on the surface properties of the
biomaterial the prekallikrein and high-molecular weight kininogen system
accelerate FXII activation. Downstream, this results in activation of Factor
XI (FXIa), Factor IX (FIXa) and Factor X (FXa). Finally, FXa activates
prothrombin to thrombin, which converts fibrinogen into fibrin to form a
fibrin meshwork.
Thrombin will also activate platelets, Factor VIII (FVIIIa) and Factor V
(FVa). Activated platelets, which expose negatively charged phospholipid,
further enhance coagulation by supporting the formation of FIXa-FVIIIa
and Fxa-FVa complexes that dramatically increase the rate of thrombin
generation. After initial fibrin clot formation, ongoing thrombin generation
is required to sustain and stabilize the clot.
Principle of the Test
The TGA gives an indication of the thrombogenicity of a biomaterial. The
amount and speed of thrombin formation, when the biomaterial is
incubated in modified plasma, is measured and compared to that of
reference materials. Highly reactive and less-reactive reference materials
are included in the kit, one type of metal and two types of polymers. It is
2
recommended to include at least two reference materials in each
analysis. Reference materials can be selected to compare with the
activities of the test samples.
Materials are first incubated in plasma. After the TGA reagent has been
added, the generation of thrombin will begin. At different time points the
thrombin generation is measured. The concentration of thrombin is
determined by enzymatic reaction with a thrombin-specific chromogenic
substrate, which yields a yellow colored product proportional to the
amount of thrombin.
The thrombin concentrations of the samples can be determined from a
calibration curve. A thrombin generation curve for each material is
constructed by plotting the thrombin concentrations versus the time
points on which the samples were taken. This curve is used to determine
the speed of thrombin generation, which is expressed per cm2
 sample.
The results of the tested materials in relation to the reference materials
may be used to evaluate the thrombogenicity. It must be noted that
pass/fail criteria cannot be applied yet since criteria for this evaluation
method are not available.
The kit is designed to determine thrombin-generating activity of small
samples of biomaterial. Larger samples can also be used as long as the
ratio between TGA plasma and material size is respected.
Precautions
• The kit is intended for research use only.
• The kit should not be used beyond its expiration date.
• Do not combine reagents from TGA Kits with different lot
numbers.
• The plasma is of human origin and has been tested and confirmed
negative for HbsAg, HCV, HIV I/II, HTLVI/II and Treponema.
However, the plasma should always be treated as a potential
biohazard during use and for disposal.
• Chemicals and reagents have to be treated as hazardous waste
according to biohazard safety guidelines or regulations. For
information on hazardous substances included in the kit please
refer to the Material Safety Data Sheets, which are available upon
request.
• Wear disposable (latex) gloves when handling specimens and
reagents.
• Never pipette by mouth and avoid contact of skin and mucous
membranes with reagents containing sodium azide, the Substrate
and Stop Solution. Wash thoroughly with large amount of water if
3
contact occurs.
• Use disposable pipette tips throughout the procedure to avoid
contamination of reagents.
Contents of the Kit
• Thrombin, 7.5 Units 75 μL 2 vials
• Buffer A, Phosphate buffer 11 mL 1 bottle 
with BSA, 0.01% sodium azide
• TGA Plasma, modified 750 μL 5 vials 
human plasma
• Saline, NaCl solution, 5 mL 1 bottle
0.01% sodium azide
• TGA Reagent A, contains 5 mL 2 bottles
0.01% sodium azide
• TGA Reagent B, contains 600 μL 2 vials
0.01% sodium azide
• Buffer B, Tris buffer, 30 mL 2 bottles 
0.01% sodium azide
• Substrate, Thrombin-specific 0.6 mL 2 vials 
chromogenic substrate
• Stop Solution, Acetic acid (20%) 6 mL 1 bottle
• Reference 1, Low-density 0,6 cm2 5 pcs
polyethylene (LDPE)
• Reference 2, Polydimethyl- 0,7 cm2 5 pcs 
siloxane (PDMS)
• Reference 3, Medical steel (MS) 0,6 cm2 5 pcs
Additional Materials and Equipment
The following materials and equipment are required but are not provided
with the kit:
• (Calibrated) adjustable pipettes with disposable tips.
• Water bath at 37 °C.
• Incubator at 37 °C.
• Timer.
• Crushed ice.
• Plate reader capable of measuring at 405 and 540 nm
wavelengths.
• Tweezers.
• Mixer.
• Micro-centrifuge vials (1,5 mL), test tubes (10 mL)
4
• Optional, for cleaning of test samples:
◦ RBS (chlorinated trisodium phosphate, sodium metasilicate) or
a comparable detergent.
◦ Sonicator.
◦ Ultra pure water.
Test Procedure
Preparation of Materials for Testing
The TGA test can be used for coated or uncoated biomaterials. It is
recommended to test clean samples. The following procedure is
recommended to clean biomaterials:
1. Sonicate the biomaterial for 15 min in 2% RBS and wash three
times with ultra pure water.
2. Incubate the biomaterial for 5 min in 70% ethanol and wash three
times with ultra pure water.
3. Dry the material in the air.
NOTES:
• Cleaned materials should always be handled with tweezers.
• This cleaning procedure is a recommendation only; each user
should determine an own optimal procedure.
• The reference materials provided in the kit are clean and ready for
use.
Reagent Preparation
Select the required reagents and number of multi-well strips. The
volumes given in the procedure are based on biomaterial pieces of 0,5 x
0,5 cm (surface area approximately 0,6 cm2
).
Keep Thrombin and TGA reagent B on ice until use. Allow all other
reagents to reach room temperature prior to use.
After first use, Buffer A, Buffer B, Saline and Stop Solution can be stored
at 2-8 °C.
Prepare reagents as follows:
• TGA Plasma: Dilute TGA plasma by adding 750 μL Saline per vial
of TGA plasma, while it is thawing. Mix gently; do not shake. After
reconstitution the TGA plasma must be stored on ice and used
within 0,5 h. Each vial is sufficient for 4 biomaterial samples.
• Substrate solution: Add 2,4 mL of Buffer B to each vial of
5
substrate. Prepare freshly prior to use. The volume per vial is
sufficient for 48 wells of a 96-well microtitre plate.
• Thrombin calibrators: Dilute 20 μL of Thrombin with 1980 μL
Buffer A in a separate vial. Use the latter dilution to prepare
Thrombin calibrators according to Table 1. First pipette Buffer A
into the vials and then the volume of diluted Thrombin. Use Buffer
A as a blank (BLK). Prepare calibrators freshly each day and store
on ice till further use.
Table 1. Preparation of Thrombin Calibrators.
Assay Procedure
NOTES IN ADVANCE:
• Never use pipettes or vials of glass since glass is a material that
may substantially activate the clotting system.
• Select 2 or 3 types of reference materials to which the activities of
the test materials can be compared. As a guideline Reference 1
(LDPE) should be considered as minimally reactive, Reference 2
(PDMS) as intermediary reactive and Reference 3 (MS) as highly
reactive.
• The reference materials provided in the kit have been cleaned by
the protocol described above and are ready for use.
• The volumes given in this procedure are based on pieces of
material of 0,5 x 0,5 cm (surface area approximately 0,6 cm2
).
Test materials should be prepared in pieces of approximately the
same size. A correction for surface area is made in the final
calculations.
• It is important to take samples at the exact time points as
described below. Since samples have to be taken at timed
intervals it is recommended that materials and controls be
incubated as described below in sets of no more than two with a
30-sec interval.
6
Thrombin C oncentration [mU/mL] Buffer A [µL] Diluted Thrombin [µL]
BLK 0 1000 0
C AL1 25 975 25
C AL2 50 950 50
C AL3 100 900 100
C AL4 150 850 150
C AL5 200 800 200
C AL6 300 700 300
C AL7 400 600 400
1. Place the selected reference and (cleaned) test materials in micro￾centrifuge vials using tweezers. Fix the materials vertically
between the walls of the vials to prevent floating. Use vials
without material as a negative control (CTRL).
2. Add 350 μL (diluted) TGA plasma to each vial with specimen (test
material, reference material or negative control). Be sure that the
biomaterial is completely immersed in the plasma.
3. Incubate the vial(s) for 15 min in a water bath at 37 °C.
4. Directly after starting the TGA plasma incubation, also incubate a
vial containing 450 μL of TGA Reagent A + 50 μL of TGA Reagent
B.
5. During the 15 min incubation of the TGA plasma and the Reagent
A + Reagent B mixture, prepare for each specimen (test material,
reference material or negative control) 4 labeled micro-centrifuge
vials (SPL1-4), each containing 490 μL Buffer B and place the vials
on ice.
6. Add, at t= 15 min, 150 μL of the mixture of TGA Reagent A and B
to the vial with plasma and mix gently.
7. At t= 16 min, collect a sample of 10 μL out of the reaction mixture
into the vial labeled SPL1, which already contains 490 μL Buffer B,
vortex and store on ice (t= 1 min). Mix the vials with reaction
medium just before removing the 10μL samples and immediately
place the vials back into the water bath after sampling.
8. Repeat this sampling procedure at t= 17 min, t= 18 min and t=
19 min to collect SPL2, SPL3 and SPL4, respectively.
9. Keep all diluted samples on ice until all test materials, reference
materials and controls have been processed. Samples can be
stored on ice for up to 2 h. For each additional material or control,
start again at step no 2.
10.Remove all calibrators and diluted samples collected in Buffer B
from the ice, vortex and place at room temperature.
11. Transfer 150 μL of each calibrator and diluted sample to the 96-
well plate. It is recommended to pipette each sample in duplicate.
Table 2 illustrates a template that might be used.
12. Incubate the plate for 2 min in an incubator at 37 °C.
13.Add 50 μL of diluted Substrate solution to each well (use of a
repetition pipette is highly recommended; pipette in the same
order as initially used to fill the plate).
14.Cover the plate with a plate cover or foil and incubate for 20 min
at 37 °C.
15. Following the incubation, add 50 μL of Stop Solution to each well.
Stop Solution should be added to the wells in the same sequence
as the Substrate was added (use of a repetition pipette is highly
recommended).
7
16.Read the optical density at 405 nm (OD405) and 540 nm
(OD540). The reading has to be performed within 30 min after the
Stop Solution was added.
17.Calculate the thrombin concentration of all diluted samples and
calculate the velocity of thrombin generation for each test
material, reference material and negative control (see
Calculations).
Table 2. Suggested 96-well template for the TGA assay.
Calculations*
1. Plot OD405 (corrected for OD540) against the thrombin
concentrations of the calibrators. The calibration curve must be a
straight line (OD405 = a x CAL + b).
2. Interpolate the thrombin concentration of all samples for all
sampling time points.
3. Construct for all test and reference materials and all controls a
thrombin generation curve, in which the concentration of thrombin
(mU/mL) is plotted against the incubation time.
4. Correct, for all test and reference materials, the thrombin
concentration at each time point for the corresponding thrombin
concentration of the negative control(s).
5. Determine for the test and reference materials and the negative
control the highest velocity of thrombin generation (ΔmU/mL/min)
in between 2 measured time points (i.e., at the point of the
steepest slope on the curve) and correct this value for the dilution
factor (50x).
8
1 2 3 4 5 6 7 8 9 10 11 12
A BLK BLK
B
C
D
E
F
G
H
C TRL
SPL1
C TRL
SPL1
REF2
SPL1
REF2
SPL1
TST2
SPL1
TST2
SPL1
TST4
SPL1
TST4
SPL1
TST6
SPL1
TST6
SPL1
C AL
1
C AL
1
C TRL
SPL2
C TRL
SPL2
REF2
SPL2
REF2
SPL2
TST2
SPL2
TST2
SPL2
TST4
SPL2
TST4
SPL2
TST6
SPL2
TST6
SPL2
C AL
2
C AL
2
C TRL
SPL3
C TRL
SPL3
REF2
SPL3
REF2
SPL3
TST2
SPL3
TST2
SPL3
TST4
SPL3
TST4
SPL3
TST6
SPL3
TST6
SPL3
C AL
3
C AL
3
C TRL
SPL4
C TRL
SPL4
REF2
SPL4
REF2
SPL4
TST2
SPL4
TST2
SPL4
TST4
SPL4
TST4
SPL4
TST6
SPL4
TST6
SPL4
C AL
4
C AL
4
REF1
SPL1
REF1
SPL1
TST1
SPL1
TST1
SPL1
TST3
SPL1
TST3
SPL1
TST5
SPL1
TST5
SPL1
TST7
SPL1
TST7
SPL1
C AL
5
C AL
5
REF1
SPL2
REF1
SPL2
TST1
SPL2
TST1
SPL2
TST3
SPL2
TST3
SPL2
TST5
SPL2
TST5
SPL2
TST7
SPL2
TST7
SPL2
C AL
6
C AL
6
REF1
SPL3
REF1
SPL3
TST1
SPL3
TST1
SPL3
TST3
SPL3
TST3
SPL3
TST5
SPL3
TST5
SPL3
TST7
SPL3
TST7
SPL3
C AL
7
C AL
7
REF1
SPL4
REF1
SPL4
TST1
SPL4
TST1
SPL4
TST3
SPL4
TST3
SPL4
TST5
SPL4
TST5
SPL4
TST7
SPL4
TST7
SPL4
6. Calculate the thrombin generation of the biomaterials and
reference materials in ΔmU/mL/min/cm2
. Reference 1 has a
surface area of 0,6 cm2
, Reference 2 of 0,7 cm2
 and Reference 3 of
0,6 cm2
.
*Upon request an electronic file can be provided to perform the
calculations.
Assay Criteria
• The correlation coefficient of the calibration curve should be
≥0.98.
• The parameters a and b of the calibration curve must be in
between the values as given in the ‘Certificate of Analysis’
(enclosed with the kit).
• The result of the negative control should be ≤2000 ΔmU/mL/min.
• The results of the reference materials should be in the ranges as
provided in the ‘Certificate of Analysis’.
• Repeat the TGA procedure if the results do not meet the assay
criteria.
Characteristics
Figure 1. Example of a thrombin calibration curve. This curve is an
example only and should not be used for calculations.
9
0 50 100 150 200 250 300 350 400 450
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
R² = 1.00
Thrombin (mU/mL)
O
D* (4
0
5
n
m - 5
4
0
n
m)
Figure 2. Typical thrombin generation curves for low-density polyethylene
(LDPE), polydimethylsiloxane (PDMS) and medical steel (MS).
Figure 3. Typical maximum thrombin generation patterns for Low-density
polyethylene (LDPE), polydimethylsiloxane (PDMS) and medical steel
(MS).
10
1 2 3 4
-500
0
500
1000
1500
2000
2500
3000
3500
4000
Thrombin Generation over time
LDPE
PDMS
MS
Time (min)
G
e
n
erate
d thro
m
bin (m
U/m
L)
LDPE PDMS MS
0
500
1000
1500
2000
2500
3000
3500
4000
4500
Maximum thrombin generation
M
a
xim
u
m thro
m
bin
g
e
n
eratio
n
(m
U/m
L/min/c
m
^
2)

Thrombin Generation Kit Insert February HAEMOSCAN BV

Thrombin Generation Kit Insert February HAEMOSCAN BV

Thrombin Generation Kit Insert February HAEMOSCAN BV

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