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  文件名称:  Biomaterial Hemolytic Assay Kit Insert November HAEMOSCAN BV
  公司名称:  上海拜力生物科技有限公司
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Biomaterial Hemolytic Assay Kit Insert November HAEMOSCAN BV

 COMPANY WITH
 QUALITY MANAGEMENT SYSTEM
 CERTIFIED BY DNV GL
 = ISO 9001 =
 Biomaterial Hemolytic
 Assay
 Kit Insert
 REVISED VERSION: November 2017
Summary
Hemolytic activity is a requirement to be tested for any blood contacting
medical device. The test is based on erythrocyte lysis induced by contact,
leachables, toxins, metal ions, surface charge or any other cause of
erythrocyte lysis. The current description is based on direct contact of
biomaterial and an erythrocyte suspension. The method is based on release
of hemoglobin, which can be measured spectrophotometrically. This method
is suited to evaluate the haemocompatibility of biomaterials and medical
devices according to the international standard ISO 10993-4:2002.
Introduction
Interactions between blood and biomaterials may induce erythrocyte lysis.
Particularly during prolonged contact or during contact of blood with large
surfaces, this so called process of hemolysis may induce adverse events, due
to anemia and the release of iron and other bioactive substances. Optimal
conditions to perform a hemolysis assay have been described. Slightly diluted
human blood in a blood/material ratio of 1 mL/cm2
 in a rotating chamber is
recommended for performing the hemolysis assay. 
Principle of the Test
An erythrocyte suspension is incubated for 24 hours with test material during
rotation at 37ºC. Before and after incubation samples are collected and
centrifuged to obtain supernatant, containing free hemoglobin. The
hemoglobin concentration is measured by means of a spectrophotometer.
Test samples are compared to reference materials. Positive reactive and less￾reactive reference materials are included in the kit. It is recommended to
include at least two reference materials in each analysis. 
The results of the tested materials in relation to the reference materials may
be used to evaluate the hemolytic activity. It must be noted that pass/ fail
criteria are based on 2% hemolysis. Thus, also the total hemoglobin
concentration of the used erythrocyte suspension must be determined.
The kit is designed to determine hemolytic activity of small biomaterial
samples. Larger samples can also be used as long as the ratio between
erythrocyte suspension and material size is respected.
2
Precautions
• Keep the kit or at least the erythrocytes in an as cold as possible
freezer. The shelf life of 6 months is based on storage at -80ºC.
• Once the erythrocytes are thawed they should not be refrozen, but be
kept at 2-8ºC. Other reagents can be frozen again.
• The kit is intended for research use only.
• The kit should not be used beyond its expiry date.
• Do not combine reagents from Hemolysis Kits with different lot
numbers.
• The erythrocytes are of human origin and have been tested and
confirmed negative for HbsAg, HCV, HIV I/II, HTLVI/II and
Treponema. However, blood 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. 
• Wear disposable (latex) gloves when handling specimens and
reagents.
• Never pipette by mouth and avoid contact of skin and mucous
membranes 
• Use disposable pipette tips throughout the procedure to avoid
contamination of reagents.
Contents of the Kit
• Erythrocyte concentrate 3 mL 3 tubes
• Wash Buffer 30 mL 1 bottle
• Dilution Buffer I 20 mL 1 bottle
• Dilution Buffer II 20 mL 1 bottle
• Dilution Buffer III 40 mL 1 bottle
• Lysis fluid 8 mL 1 vial
• Hemoglobin (10 mg/mL) 0.5 mL 1 bottle
• Assay Buffer 10 mL 1 bottle
• Reference 1, Silicon elastomer (SE) 0.5 cm2 5 pcs 
• Reference 2, Buna N 0.5 cm2 5 pcs 
Reference 3, Medical steel (MS) 0.5 cm2 5 pcs 
• RBS cleaning solution 2 mL 1 vial
(chlorinated trisodium phosphate, sodium metasilicate) 
Additional Materials and Equipment
The following materials and equipment are required but are not provided with
the kit:
• (Calibrated) adjustable pipettes with disposable tips.
• Incubator at 37°C.
3
• Spectrophotometer capable of measuring at 415, 450, and 380 nm
• Tweezers.
• Micro-centrifuge + vials (1.5 mL).
• Optional, for cleaning of test samples: Sonicator.
• Ultra pure water.
• Ethanol (70%).
Test Procedure
Preparation of Materials for Testing
The Hemolysis test can be used for coated or uncoated biomaterials. It might
be important that clean samples be tested. The following procedure is
recommended to clean biomaterials without coating:
1. Dilute RBS 20 times in UP water.
2. Sonicate the biomaterial for 15 min in the 5% RBS solution and wash
three times with ultra pure water.
3. Sonicate the biomaterial for 5 min in 70% ethanol and wash three
times with ultra pure water.
4. Dry the material in the air.
Notes:
• Cleaned materials should always be handled with clean tweezers.
• This cleaning procedure is a recommendation only; each user should
determine his or her own optimal procedure. This procedure may
affect a (biological) coating.
• The reference materials provided in the kit are clean and ready for
use.
Reagent Preparation
Select the required reagents and number of bottles to be used. Keep spare
erythrocyte tubes between -80ºC and -20ºC. The volumes given in the
procedure are based on biomaterial pieces of 0.5 x 0.5 cm (surface area 2-
sided approximately 0.5 cm2
).
Allow all reagents to obtain room temperature.
Prepare reagents as follows:
• Erythrocyte suspension: Add 5 mL Wash Buffer to the erythrocyte
suspension. Mix gently by end-over-end tumbling of the tube.
Centrifuge the tube, for 10 minutes at 1200xg. Remove the
supernatant. Repeat this procedure. Then slowly add 5 ml Dilution
buffer I mix gently, centrifuge, and perform the same wash procedure
once with 5 mL Dilution Buffer II and once with Dilution Buffer III.
The pellet is than resuspended in 5 mL Dilution buffer III. (The quality
of the final suspension is controlled by centrifuging 0.5 mL in an
4
eppendorf, which should result in an almost colorless supernatant).
Each washed vial is sufficient for 10 biomaterial samples.
• Test samples: Samples are preferably placed in a 2 mL syringe.
After the erythrocyte suspension has been added, the remaining air is
removed from the syringe, which is capped or closed with parafilm. In
a standard tube the suspension should obtain air bubbles as little as
possible. The negative control is a syringe without any material.
• Hemoglobin calibrators: The hemoglobin standard is used undiluted
and further stepwise (1:1) diluted with Lysis fluid in separate vials.
Use these dilutions as standard curve (Table 1).
Table 1. Preparation of Hemoglobin Calibrators.
Hemoglobin
Concentration
(mg/mL)
CAL1 350 10
CAL2 150 Cal 1 + 150 lysis fluid 5
CAL3 150 Cal 2 + 150 lysis fluid 2,5
CAL4 150 Cal 3 + 150 lysis fluid 1,25
CAL5 150 Cal 4 + 150 lysis fluid 0,63
CAL6 150 Cal 5 + 150 lysis fluid 0,31
CAL7 150 Cal 6 + 150 lysis fluid 0,15
BLK 150 lysis fluid 0
Assay Procedure
Notes in Advance:
■ 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 (2-sided: surface area approximately 0.5 cm2
). Larger
materials need more volume. Test materials should be prepared in pieces
of approximately the same size. A correction for surface area is made in
the final calculations.
■ Always avoid intense air contact and bubbles, since this may increase
Hemolysis.
1. Place the selected reference and (cleaned) test materials in syringes
or tubes using tweezers. Use syringes without material as negative
control (CTRL).
2. Add 0.5 mL erythrocyte suspension to each vial with specimen (test
material, reference material or negative control). Be sure that the
biomaterial is completely immersed in the suspension. A syringe is
closed by the plunger, air is removed and a cap or parafilm is used to
close the outlet.
5
3. Add 10 µL of erythrocyte suspension to 990 µL of lysis fluid, to be
used as total hemoglobin concentration.
4. Incubate the vials end-over end rotating at 37°C for 24 hours.
5. Transfer the erythrocyte suspension to a small vial (such as
eppendorf), take care to avoid bubbles.
6. Centrifuge the vials at high speed (>4000xg) for 1 minute.
7. Add 20 µL of supernatant from sample or standard to a well of a
microtiter plate.
8. Add 180 µL Assay Buffer.
9. Mix on a plate shaker.
10. Measure OD at 415/450/380 nm (Harboe method) or OD 415 if the
other filters are not available. The calculated OD* from the Harboe
method is (2 x 415) – (450 + 380).
Calculations*
1. Plot the calculated OD* against the hemoglobin concentrations of the
calibrators. The calibration curve should be a straight line (OD*= a x
CAL + b).
2. Correct for the hemoglobin concentration of the negative control.
3. Calculate the hemoglobin concentration of the biomaterials and
reference materials in mg/cm2

4. Calculate the hemoglobin concentration in %/cm2
 by the total
hemoglobin concentration value corrected for 100 x dilution.
Assay Criteria
• The correlation coefficient of the calibration curve should be ≥0.98.
• The result of the negative control should be ≤0.1% of total
hemoglobin.
6
Characteristics
Figure 1. Example of Hemoglobin standard curve.
Figure 2. Example of hemolysis of the reference materials, corrected for
negative control.
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0 1 2 3 4 5 6
0
1
2
3
4
5
6
Hemoglobin [mg/mL]
O
D*
SE Buna-N MS
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0,4
0,45
0,5
Hb [mg/mL]

Biomaterial Hemolytic Assay Kit Insert November HAEMOSCAN BV

Biomaterial Hemolytic Assay Kit Insert November HAEMOSCAN BV

Biomaterial Hemolytic Assay Kit Insert November HAEMOSCAN BV

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