ASTM F756-00
(Practice)Standard Practice for Assessment of Hemolytic Properties of Materials
Standard Practice for Assessment of Hemolytic Properties of Materials
SIGNIFICANCE AND USE
The presence of hemolytic material in contact with the blood may cause loss of, or damage to, red blood cells and may produce increased levels of free plasma hemoglobin capable of inducing toxic effects or other effects which may stress the kidneys or other organs.
This practice may not be predictive of events occurring during all types of implant applications. The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F 748.
SCOPE
1.1 This practice provides a protocol for the assessment of hemolytic properties of materials used in the fabrication of medical devices that will contact blood.
1.2 This practice is intended to evaluate the acute in vitro hemolytic properties of materials intended for use in contact with blood.
1.3 This practice consists of a protocol for a hemolysis test under static conditions with either an extract of the material or direct contact of the material with blood.
1.4 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide guidance for the selection of appropriate methods for testing materials for a specific application.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
1.6 Identification of a supplier of materials or reagents is for the convenience of the user and does not imply single source. Appropriate materials and reagents may be obtained from many commercial supply houses.
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Designation:F756–00
Standard Practice for
Assessment of Hemolytic Properties of Materials
This standard is issued under the fixed designation F 756; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope 3.1.2 % hemolysis—the quotient of the free plasma hemo-
globin (mg/ml) released as a result of contact with test material
1.1 This practice provides a protocol for the assessment of
or extract divided by the total hemoglobin (mg/ml) present in
hemolytic properties of materials used in the fabrication of
thebloodsolutionmultipliedby100.(Thisissynonymouswith
medical devices that will contact blood.
hemolytic index).
1.2 This practice is intended to evaluate the acute in vitro
3.1.3 comparative hemolysis—comparison of the hemolytic
hemolytic properties of materials intended for use in contact
index produced by a test material compared with that produced
with blood.
by a standard reference material such as polyethylene at the
1.3 This practice consists of a protocol for a hemolysis test
same test conditions.
under static conditions with either an extract of the material or
3.1.4 direct contact test—a test for the hemolytic property
direct contact of the material with blood.
performed with the test material in direct contact with the
1.4 This practice is one of several developed for the
blood.
assessment of the biocompatibility of materials. Practice F 748
3.1.5 extract test—a test for the hemolytic property per-
may provide guidance for the selection of appropriate methods
formed with an isotonic extract of the test material, as
for testing materials for a specific application.
described in F 619, in contact with the blood.
1.5 This standard does not purport to address all of the
3.1.6 hemolysis—destruction of erythrocytes resulting in
safety concerns, if any, associated with its use. It is the
the liberation of hemoglobin into the plasma or suspension
responsibility of the user of this standard to establish appro-
medium.
priate safety and health practices and determine the applica-
3.1.7 negative control—a material, such as a polyethylene,
bility of regulatory limitations prior to use.
that produces little or no hemolysis (<2 % after subtraction of
1.6 Identification of a supplier of materials or reagents is
the blank) in the test procedure. It is desirable that the control
for the convenience of the user and does not imply single
specimens have the same configuration as the test samples.
source. Appropriate materials and reagents may be obtained
3.1.8 positive control—a material capable of consistently
from many commercial supply houses.
producing a hemolysis of at least 8 % after subtraction of the
2. Referenced Documents results of the blank. Suggested materials include, but are not
limited to, BUNA N (Aero Rubber and other suppliers), and
2.1 ASTM Standards:
plastisol (Plasti-Coat, Watertown CT.).
F 619 Practice for Extraction of Medical Plastics
3.1.9 cyanmethemoglobin reagent—a reagent to which
F 748 Practice for Selecting Generic Biological Test Meth-
whole blood, plasma, or test supernatant is added that quickly
ods for Materials and Devices
converts most of the forms of hemoglobin to the single
3. Terminology
cyanmethemoglobin form for quantification at its 540 nm
spectrophotometric peak. The reagent (based on that by van
3.1 Definitions:
Kampen and Zijlstra (1), pH 7.0-7.4), is made with 0.14 g
3.1.1 plasma hemoglobin—the amount of hemoglobin in
potassium phosphate, 0.05 g potassium cyanide, 0.2 g potas-
the plasma.
siumferricyanide,and0.5-1mlofnonionicdetergentdilutedto
1 L with distilled water. The conversion time of this reagent is
This practice is under the jurisdiction ofASTM Committee F-4 on Medical and
3-5 minutes. This reagent is recommended by the National
Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.16 on Biocompatibility.
Commission for Clinical Laboratory Studies (NCCLS) and
Current edition approved July 10, 2000. Published October 2000. Originally
may be made from the chemicals or purchased from supply
published as F 756 – 82. Last previous edition F 756 – 93.
2 houses.
Annual Book of ASTM Standards, Vol 13.01.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F756
The first cyanmethemoglobin reagent used to measure total 6. Preparation of Test and Control Specimens
blood hemoglobin concentration was Drabkin’s Reagent (1 g
6.1 Specimenswithadeterminablesurfaceareawillbeused
of sodium bicarbonate, 0.05g of potassium cyanide, 0.2 g of
at a ratio of 3 cm surface area to 1 ml of test blood solution.
potassium ferricyanide and diluted with distilled water to 1 L).
For irregular materials or powders, a weight of 0.2 to 0.5 g per
The disadvantages of using the Drabkin’s reagent versus the
ml of liquid will be used.Aminimum of 6 of each positive and
NCCLS cyanmethemoglobin reagent are that it has a conver-
negative control and each test sample should be prepared to be
sion time of 15 minutes, and pH of 8.6 which may cause
used in the direct contact test and the test with the extract.
turbidity. However, it is still available as individual chemicals
6.2 The final sample should be prepared with a surface
or kits such as Sigma 525-A.
finish consistent with end-use application.
The Drabkin’s and cyanmethemoglobin reagents were de-
6.3 The sample shall be sterilized by the method to be
veloped to quantify the high hemoglobin concentration nor- employed for the final product.
mally found in whole blood (e.g. 15,000 mg/dl). By modifying 6.4 Care should be taken that the specimens do not become
the sample dilution volumes and accounting for background
contaminated during preparation but aseptic technique is not
interference, these reagents can also be used to measure much required.
lower plasma or supenatant hemoglobin concentrations as well
(Moore et al, Malinauskas (2), (3)).
7. Hemoglobin Determination (Direct Method)
3.1.10 PBS—phosphate buffered saline (Ca and Mg free).
7.1 Prepare a hemoglobin calibration curve consistent with
The use of phosphate buffered saline is preferable to the use of
the specifications of the International Committee for Standard-
saline in order to maintain the pH. The use of magnesium and
ization in Hematology (ICSH). For hemoglobin standardiza-
calcium free PBS is necessary to maintain the anticoagulant
tion use reference standards such as the Sigma Hemoglobin
properties of the chelating agents used in collecting the blood.
Reference Standard No 525 or equivalent and the suggested
It is used as the background or “blank” for a hemolysis test.
reagent diluent. The spectrophotometer used should be high
resolution and provide at least 3 decimal places.
3.1.11 A—absorbance value of cyanmethemoglobin reac-
tion product measured at 540 nm. 7.2 Prepare a standard curve from stock hemoglobin in 8
dilutions to accommodate the range of 1.4 to 0.03 mg/ml (140-
3.1.12 F—slope of the hemoglobin curve. The units are
3 mg/dl). The cyanmethemoglobin reagent diluent serves as a
[(mg/ml)/A] such that multiplication by an absorbance value
zero blank in the spectrophotometer. Measure the absorbance
yields a hemoglobin concentration. Implicit assumption: The
at 540 nm. Plot a calibration curve from these values using
y-intercept of the hemoglobin calibration curve is approxi-
mg/ml Hb on the y axis andA on the x-axis. The calibration
mately zero and its effect on converting absorbance values to 540
coefficient (F) is the slope of this plot. The y-intercept should
concentration values is negligible.
be approximately zero.
3.1.13 PFH—plasma free hemoglobin concentration.
NOTE 1—If local restrictions or other problems contraindicate use of
3.1.14 C—total blood hemoglobin concentration.
these cyanmethemoglobin reagents, then another method for measuring
3.1.15 T—diluted blood hemoglobin concentration.
total blood hemoglobin concentration, plasma free hemoglobin concen-
3.1.16 B—blank(i.e.nomaterialaddedtothistube,onlythe
tration, and supernatant hemoglobin concentration may be substituted
isotonic medium).
providedthatitisvalidatedandshowntobesubstantiallyequivalenttothe
cyanmethemoglobin method. Methods which quantify oxyhemoglobin
3.1.17 S—sample (i.e. test material sample, or negative and
alone may not be appropriate since some materials can convert oxyhemo-
positive control sample).
globin to other forms or alter the absorbance spectrum. Investigators
should be aware that their results of determining supernatant hemoglobin
4. Summary of Practice
concentration may be compromised by absorption of hemoglobin by the
test materials, precipitation of hemoglobin out of solution, or alteration of
4.1 Test and control material specimens or extracts are
the spectrophotometric absorbance spectrum by material leachables.
exposed to contact with rabbit blood under defined static
conditions and the increase in released hemoglobin is mea-
8. Collection and Preparation of Blood Substrates
sured. Comparisons are made with the control and test speci-
8.1 Obtain anti-coagulated rabbit blood from at least 3
mens tested under identical conditions.
donors for each test day. The preferred anticoagulant is citrate
(0.13 M). Approximately 5 ml should be drawn from each
5. Significance and Use
rabbit. Store the blood at 4 6 2° C and preferably use within
5.1 The presence of hemolytic material in contact with the
48 h. Blood may be used up to 96 h after collection if the
bloodmaycauselossof,ordamageto,redbloodcellsandmay
plasma free hemoglobin is not excessive. Equal quantities of
produce increased levels of free plasma hemoglobin capable of
blood from each rabbit should be pooled.
inducing toxic effects or other effects which may stress the
8.2 Do not wash cells; use them suspended in the original
kidneys or other organs.
plasma.
5.2 This practice may not be predictive of events occurring 8.3 Determination of Plasma Free Hemoglobin (PFH):
8.3.1 Centrifuge a 3-ml sample of the pooled blood at
during all types of implant applications. The user is cautioned
to consider the appropriateness of the method in view of the 700-800 G in a standard clinical centrifuge for 15 min.
materials being tested, their potential applications, and the 8.3.2 Add 0.5 ml of plasma to 0.5 ml of cyanmethemoglo-
recommendations contained in Practice F 748. bin solution or validated diluent.
F756
8.3.3 Read the absorbance of the resulting solution at 540 individual tubes as described in 9.1.3. For samples with a
nm.Obtaintheconcentrationfromthestandardcurve.Multiply determinablesurfacearea,thesurfaceareashouldbe21cm or
by 2 to obtain, and record, the total plasma free hemoglobin 1.4 to 3.5 g if the specimen is irregular or a powder. The
concentration (PFH), although it has not been corrected for the recommended tube size is 16 x 125 mm. However the tube size
plasma background interference. Plasma free hemoglobin (mg/ may be any such that the specimen is covered by 7 ml of PBS
ml): liquid. Place 7 ml of PBS into each tube containing the
nonextracted sample. Place 7 ml of PBS into each of 3 tubes to
PFH
PFH 5 A *F*2 (1)
serve as the blank.
8.3.4 Proceed with the testing if the value of the PFH is less
9.3 Test—Add 1 ml of blood prepared in 8.4.4 to each tube
than 2 mg/ml. If the PFH is 2mg/ml or greater this sample
containing extract, each tube containing a specimen, and the
should be discarded and another blood sample should be
blanks. Cap all tubes.
obtained.
NOTE 3—This procedure calls for preparing the sample, adding the
8.4 Determination of Total Blood Hemoglobin Concentra-
diluent to the sample and then adding the blood which minimizes the time
tion:
difference for contact of sample with blood. Alternatively, the blood may
8.4.1 Add 20 µL of well-mixed pooled whole blood speci-
beaddedtothediluentandthenthesampleaddedtothepreparedsolution.
men to 5.0 ml of cyanmethemoglobin solution or validated
Whichever method is chosen must be used for the controls as well as the
diluent.
test specimens.
8.4.2 Allow the resulting solution to stand 15 minutes for
9.4 Maintain tubes in a suitable test tube rack for at least 3
Drabkins or 5 min for cyanmethemoglobin reagent and then
hat37° 6 2 C in a water bath. Gently invert each tube twice
readtheabsorbanceofthesolutionwithaspectrophotometerat
approximately every 30 min to maintain contact of the blood
a wavelength of 540 nm.
and material. In some cases of samples with complicated
8.4.3 Determine blood hemoglobin concentration from the
configurations, it may be necessary to do more inversions to
standard curve and multiply by 251 to account for dilution.
adequately mix the sample.
Thisshouldbeperformedinduplicate.Totalbloodhemoglobin
9.5 At the end of the specified incubation time, transfer the
concentration:
fluid to a suitable tube and centrifuge at 700-800 G for 15 min
C
C 5 A *F*251 (2)
in a standard clinical centrifuge.
8.4.4 Adjust the total hemoglobin content of the blood 9.6 Remove the supernatant carefully to avoid disturbing
sample to 10 mg/ml 6 l mg/ml by diluting with an appropriate any button of erythrocytes which may be present. Place the
amount of calcium and magnesium free PBS. Verify the supernatant into a second screw cap tube. Record the presence
hemoglobin concentration by repeating 8.4.1-8.4.3 in triplicate
of any color to the supernatant and any precipitate.
but using 400 µl of the diluted blood to 5 ml of reagent to
9.7 Analyze the samples from 9.6 for supernatant hemoglo-
remain on the standard curve. This is a dilution factor of 13.5.
bin concentration using the method in 9.8.
T
9.8 Supernatant Hemoglobin Determination:
T 5 A *F*13.5 (3)
9.8.1 Add 1.0 ml of supernatant to 1.0 ml of cyanmethemo-
globin reagent, or validated diluent.
9. Procedure for the Test
9.8.2 Allow the sample to stand for 15 min for Drabkin’s or
9.1 Extract:
3-5 min for cyanmethemoglobin reagent. Read the absorbance
9.1.1 Prepare an extract of each of 3 replicate samples of
of the solution with a spectrophotometer at a wavelength of
each test, positive control, negative control material, and PBS
540 nm.
blankaccordingtoPracticeF 619usingtheappropriateratioof
9.8.3 In the unlikely event that A exceeds 2, dilute the 1
material to extractant. (The extractant is Mg Ca free PBS). A
2 ml of the supernatant with 3 ml of diluent and repeat 9.8.
sample of 30 cm surface area or 2-5 gram is recommended for
9.8.3.1 Determine the hemoglobin concentration in each
use in 10 ml of extractant.
super
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