ASTM F756-17

Standard Practice for Assessment of Hemolytic Properties of Materials

SIGNIFICANCE AND USE
5.1 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.  
5.2 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 F748.
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. It is recommended that both tests (extract and direct contact) be performed unless the material application or contact time justifies the exclusion of one of the tests.  
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. Test Method E2524 provides a protocol using reduced test volumes to assess the hemolytic properties of blood-contacting nanoparticulate materials; this may include nanoparticles that become unbound from material surfaces.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 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.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 28-Feb-2017
• ICS: 11.100 - Laboratory medicine
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.16 - Biocompatibility Test Methods

Relations

• Replaces: ASTM F756-13 - Standard Practice for Assessment of Hemolytic Properties of Materials
• Effective Date: 01-Mar-2017
• Refers: ASTM F748-16 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Apr-2016
• Refers: ASTM E691-13 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-May-2013
• Refers: ASTM E691-11 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Nov-2011
• Refers: ASTM F748-06(2010) - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Jun-2010
• Refers: ASTM E691-08 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Oct-2008
• Refers: ASTM F619-03(2008) - Standard Practice for Extraction of Medical Plastics
• Effective Date: 01-Aug-2008
• Refers: ASTM E2524-08 - Standard Test Method for Analysis of Hemolytic Properties of Nanoparticles
• Effective Date: 01-Feb-2008
• Refers: ASTM F748-06 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Dec-2006
• Refers: ASTM E691-05 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Nov-2005
• Refers: ASTM F748-04 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-May-2004
• Refers: ASTM F619-03 - Standard Practice for Extraction of Medical Plastics
• Effective Date: 01-Nov-2003
• Refers: ASTM F619-02 - Standard Practice for Extraction of Medical Plastics
• Effective Date: 10-Apr-2002
• Refers: ASTM E691-99 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 10-May-1999
• Refers: ASTM F748-98 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 10-Aug-1998

Overview

ASTM F756-17 - Standard Practice for Assessment of Hemolytic Properties of Materials establishes a widely recognized protocol for evaluating the blood compatibility of materials, particularly focused on their potential to induce hemolysis. Hemolysis refers to the destruction of red blood cells, resulting in the release of hemoglobin into plasma, which can lead to toxic effects and stress on organs such as the kidneys. This standard is essential for manufacturers and researchers working with medical devices and materials that will come into contact with blood.

ASTM F756-17 provides practical, reproducible in vitro methods to determine if materials are hemolytic, nonhemolytic, or slightly hemolytic. The assessment is critical for minimizing adverse biological reactions in blood-contacting medical devices, contributing to patient safety and regulatory compliance.

Key Topics

• Purpose and Scope

  • The standard outlines procedures to assess the acute in vitro hemolytic properties of materials intended for blood contact.
  • It is suitable for materials used in the fabrication of medical devices, such as catheters, vascular grafts, and blood storage containers.

• Testing Protocols

  • Two main test types are described:
    • Direct contact of material with blood
    • Extract test where an extract of the material is brought into contact with blood
  • Both tests are recommended, unless justified otherwise by specific application or exposure time.

• Controls and Reference Materials

  • Tests are performed with negative controls (materials known to be nonhemolytic) and positive controls (materials known to be hemolytic) to validate results.
  • The outcome, called the hemolytic index (% hemolysis), is compared to these controls.

• Reporting Requirements

  • The standard specifies detailed reporting on sample preparation, blood characteristics, testing conditions, and test results, including mean and standard deviation of hemolytic indices.

• Limitations

  • The test may not reflect all types of in vivo responses, especially for long-term implantable devices. Users should consult ASTM F748 for guidance on method selection.

Applications

ASTM F756-17 is primarily applied in the medical device industry for critical biocompatibility assessment during product development and preclinical testing, including:

• Material screening: Early-stage evaluation of polymers, metals, and other materials for use in blood-contacting devices.
• Product validation: Demonstrating compliance with regulatory requirements for devices such as intravenous catheters, extracorporeal circuits, dialysis membranes, vascular stents, and cardiac valves.
• Quality assurance: Routine quality control to ensure ongoing material safety and performance in manufacturing.

Using ASTM F756-17 helps organizations:

• Reduce the risk of adverse hemolytic reactions in patients.
• Facilitate regulatory submissions and approval by meeting international standards for blood compatibility.
• Compare the hemolytic potential of new or alternative materials against established benchmarks.

Related Standards

ASTM F756-17 references and complements several other important standards in the assessment of medical device biocompatibility:

• ASTM F748 - Guide for selecting generic biological test methods for materials and devices, offering broader context for method selection.
• ASTM F619 - Practice for extraction of medical plastics, relevant for preparing extracts in the hemolysis test.
• ASTM E691 - Practice for conducting interlaboratory studies to determine the precision of test methods, supporting reproducibility.
• ASTM E2524 - Test method for analysis of hemolytic properties of nanoparticles using reduced test volumes, especially for blood-contacting nanomaterials.

Summary

ASTM F756-17 provides a standardized, internationally recognized protocol for evaluating the hemolytic properties of materials in medical devices, ensuring they are suitable for blood contact. Following this standard promotes patient safety, supports regulatory compliance, and assists in the rigorous assessment of blood compatibility, which is vital for the successful development and application of medical technologies.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: F756 − 17
Standard Practice for
Assessment of Hemolytic Properties of Materials
ThisstandardisissuedunderthefixeddesignationF756;thenumberimmediatelyfollowingthedesignationindicatestheyearoforiginal
adoptionor,inthecaseofrevision,theyearoflastrevision.Anumberinparenthesesindicatestheyearoflastreapproval.Asuperscript
epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
1.1 This practice provides a protocol for the assessment of 2.1 ASTM Standards:
hemolytic properties of materials used in the fabrication of E691Practice for Conducting an Interlaboratory Study to
medical devices that will contact blood. Determine the Precision of a Test Method
E2524Test Method forAnalysis of Hemolytic Properties of
1.2 This practice is intended to evaluate the acute in vitro
Nanoparticles
hemolytic properties of materials intended for use in contact
F619Practice for Extraction of Medical Plastics
with blood.
F748PracticeforSelectingGenericBiologicalTestMethods
1.3 This practice consists of a protocol for a hemolysis test
for Materials and Devices
under static conditions with either an extract of the material or
direct contact of the material with blood. It is recommended
3. Terminology
that both tests (extract and direct contact) be performed unless
3.1 Definitions of Terms Specific to This Standard:
the material application or contact time justifies the exclusion
3.1.1 plasma hemoglobin—amount of hemoglobin in the
of one of the tests.
plasma.
1.4 This practice is one of several developed for the
3.1.2 % hemolysis—free plasma hemoglobin concentration
assessment of the biocompatibility of materials. Practice F748
(mg/mL) divided by the total hemoglobin concentration (mg/
may provide guidance for the selection of appropriate methods
mL) present multiplied by 100. This is synonymous with
for testing materials for a specific application. Test Method
hemolytic index.
E2524providesaprotocolusingreducedtestvolumestoassess
3.1.3 comparative hemolysis—comparison of the hemolytic
the hemolytic properties of blood-contacting nanoparticulate
index produced by a test material with that produced by a
materials;thismayincludenanoparticlesthatbecomeunbound
standard reference material such as polyethylene under the
from material surfaces.
same test conditions.
1.5 The values stated in SI units are to be regarded as
3.1.4 direct contact test—testforhemolysisperformedwith
standard. No other units of measurement are included in this
the test material in direct contact with the blood.
standard.
3.1.5 extract test—test for hemolysis performed with an
1.6 This standard does not purport to address all of the
isotonic extract of the test material in contact with blood, as
safety concerns, if any, associated with its use. It is the
described in Practice F619.
responsibility of the user of this standard to establish appro-
3.1.6 hemolysis—destructionoferythrocytesresultinginthe
priate safety and health practices and determine the applica-
liberation of hemoglobin into the plasma or suspension me-
bility of regulatory limitations prior to use.
dium.
1.7 This international standard was developed in accor-
dance with internationally recognized principles on standard- 3.1.7 negative control—material, such as polyethylene, that
ization established in the Decision on Principles for the produces little or no hemolysis (<2 % after subtraction of the
Development of International Standards, Guides and Recom-
blank) in the test procedure. It is desirable that the control
mendations issued by the World Trade Organization Technical specimens have the same configuration as the test samples.
Barriers to Trade (TBT) Committee.
3.1.8 positive control—materials capable of consistently
producing a hemolytic index (above the negative control) of at
least 5% (see 10.3). Although positive control materials have
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
Surgical Materials and Devicesand is the direct responsibility of Subcommittee
F04.16 on Biocompatibility Test Methods. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved March 1, 2017. Published April 2017. Originally contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
approved in 1982. Last previous edition approved in 2013 as F756–13. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/F0756-17. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F756 − 17
not been validated for this practice, washed Buna N rubber 3.1.10 PBS—phosphate buffered saline (Ca- and Mg-free).
(Aero Rubber Company; ARC-45010, 0.031 in. thick sheet) The use of phosphate buffered saline is preferable to the use of
and vinyl plastisol (Plasti-Coat; 0.025 to 0.075 in. thick sheet, saline in order to maintain the pH.The use of magnesium- and
color: DB1541-medium blue 300) produced hemolysis levels calcium-free PBS is necessary to maintain the anticoagulant
above 90% when using extracts obtained at 121°C for 1 h properties of the chelating agents used in collecting the blood.
during limited interlaboratory round robin evaluations. In It is used as the background or “blank” for a hemolysis test.
x
direct contact testing, Buna N rubber (ARC-45010) produced
3.1.11 A — absorbance value of cyanmethemoglobin reac-
hemolysis levels of 14.5 6 5.3%.
tion product measured at 540 nm, where “x” represents the
specimen in 3.1.13 – 3.1.17.
NOTE 1—The specific materials tested during the revision of this
practice are available from Aero Rubber Company or Plasti-Coat.
3.1.12 F—slope of the hemoglobin standard curve. The
However,thematerialsarenotcertifiedforthisapplication,theirshelflife
units are [(mg/mL)/A] such that multiplication by an absor-
as positive controls has not been determined, and precision per Practice
bance value yields a hemoglobin concentration. Implicit as-
E691 has not been established. Hence, all available materials may not be
sumption: The y-intercept of the hemoglobin calibration curve
suitable as positive control materials for this application. Materials
considered for use in this application shall be checked for suitability in
is approximately zero and its effect on converting absorbance
accordance with the requirements in this section. If you are aware of
values to concentration values is negligible.
positive control materials, please provide this information to ASTM
3.1.13 PFH—plasma free hemoglobin concentration.
International Headquarters.
3.1.9 cyanmethemoglobin reagent—reagent to which is 3.1.14 C—total blood hemoglobin concentration.
added whole blood, plasma, or test supernatant that quickly
3.1.15 T—diluted blood hemoglobin concentration.
converts most of the forms of hemoglobin to the single
3.1.16 B—blank(thatis,nomaterialaddedtothistube,only
cyanmethemoglobin form for quantification at its 540 nm
the isotonic medium).
spectrophotometric peak. The reagent (based on that by van
3.1.17 S—sample (that is, test material sample, or negative
Kampen and Zijlstra, pH 7.0-7.4), is made with 0.14 g
and positive control sample).
potassium phosphate, 0.05 g potassium cyanide, 0.2 g potas-
sium ferricyanide, and 0.5 to 1 mL of nonionic detergent
4. Summary of Practice
diluted to 1 Lwith distilled water. The conversion time of this
reagent is 3 to 5 min. This reagent is recommended by the
4.1 Test and control material specimens or extracts are
National Commission for Clinical Laboratory Studies (NC-
exposed to contact with rabbit blood under defined static
CLS) and may be made from the chemicals or purchased from
conditions and the increase in released hemoglobin is mea-
supply houses.
sured. Comparisons are made with the control and test speci-
3.1.9.1 Discussion—The first cyanmethemoglobin reagent
mens tested under identical conditions. It is recommended that
used to measure total blood hemoglobin concentration was
both tests (extract and direct contact) be performed unless the
Drabkin’s reagent (1 g of sodium bicarbonate, 0.05 g of
material application or contact time justifies the exclusion of
potassium cyanide, 0.2 g of potassium ferricyanide and diluted
one of the tests.
with distilled water to 1 L). The disadvantages of using the
Drabkin’s reagent compared to the NCCLS cyanmethemoglo-
5. Significance and Use
bin reagent are that it has a conversion time of 15 min and pH
5.1 The presence of hemolytic material in contact with the
of8.6,whichmaycauseturbidity.However,Drabkin’sreagent
bloodmaycauselossof,ordamageto,redbloodcellsandmay
is still available from commercial suppliers.
produceincreasedlevelsoffreeplasmahemoglobincapableof
3.1.9.2 Discussion—The Drabkin’s and cyanmethemoglo-
inducing toxic effects or other effects which may stress the
bin reagents were developed to quantify the high hemoglobin
kidneys or other organs.
concentration normally found in whole blood (for example,
5.2 This practice may not be predictive of events occurring
15000mg/dL).Bymodifyingthesampledilutionvolumesand
during all types of implant applications. The user is cautioned
accounting for background interference, these reagents can
to consider the appropriateness of the method in view of the
also be used to measure much lower plasma or supernatant
materials being tested, their potential applications, and the
hemoglobin concentrations as well (Moore et al,
6,7
recommendations contained in Practice F748.
Malinauskas).
6. Preparation of Test and Control Specimens
6.1 SamplesshouldbepreparedinaccordancewithPractice
Aero Rubber Company, 8100 W. 185th St., Tinley Park, IL 60487, http://
www.aerorubber.com; Plasti-Coat, 137 Brookside Dr., Waterbury, CT 06708,
F619. A minimum total of six positive and six negative
http://www.plasti-coat.com. See Note 1.
controls, along with six test samples, should be prepared to be
Malczewski, R, Jackson,A, Lee, M, Malinauskas, R, Merritt, K, Peterson, L.,
usedinthedirectcontacttestandthetestwiththeextract(three
“Standardizing an in vitro Hemolysis Assay for Screening Materials Used in
Medical Devices ,” Society for Biomaterials, Tampa, FL, Apr. 2002 (Extended samples per test).
abstract).
International Committee for Standardization in Haematology. J Clin. Pathol,
Vol 49, 1996, pp. 271–274.
6 7
Moore, G. L., Ledford, M. E., Merydith, A., “A micromodification of the Malinauskas, R. A., “Plasma hemoglobin measurement techniques for the in
Drabkin hemoglobin assay for measuring plasma hemoglobin in the range of 5 to vitroevaluationofblooddamagecausedbymedicaldevices,” Artificial Organs,Vol
2000 mg/dl,” Biochem. Med, Vol 26, 1981 , pp. 167–173. 21, 1997, pp. 1255–1267.
F756 − 17
6.2 The final sample should be prepared with a surface 8.3.2 Perform a 1:1 dilution of the plasma with the cyan-
finish consistent with its end-use application. methemoglobin reagent or validated diluent (for example, add
0.5 mL of plasma to 0.5 mL of cyanmethemoglobin reagent).
6.3 The sample shall be sterilized by the method to be
8.3.3 Read the absorbance of the resulting solution at 540
employed for the final product.
nm after 15 min. Obtain the concentration from the standard
6.4 Care should be taken that the specimens do not become
curve.Multiplyby2toobtain,andrecord,thetotalplasmafree
contaminated during preparation but aseptic technique is not
hemoglobin concentration (PFH), although it has not been
required.
corrected for the plasma background interference. Plasma free
hemoglobin (mg/mL) is calculated as follows:
7. Hemoglobin Determination (Direct Method)
PFH
PFH 5 A 3F 32 (1)
7.1 To create a hemoglobin concentration calibration curve
using the cyanmethemoglobin method, use commercially 8.3.4 ProceedwiththetestingifthevalueofthePFHisless
available reference standards and reagents from clinical diag-
than 2 mg/mL. If the PFH is 2mg/mL or greater, this sample
nostic companies that conform to the specifications of the should be discarded and another blood sample should be
International Committee for Standardization in Hematology
obtained.
5 8
(ICSH). OnecommercialsourceismadebyPointeScientific.
8.4 Determination of Total Blood Hemoglobin
A spectrophotometer that provides absorbance readings to at
Concentration—Note that the total blood hemoglobin concen-
least three decimal places, and is able to detect the entire
tration can be determined either by the cyanmethemoglobin
hemoglobinconcentrationrange(asspecifiedin7.2)shouldbe
method (detailed below) or by using a validated hemoglobin-
used.
ometertoreplacesteps8.4.1–8.4.3.However,afterdilutionin
7.2 Prepareastandardcurvefromasuitablestandardinsix
step 8.4.4, the total blood hemoglobin concentration may be
dilutionstoaccommodatetherangeof0.03to0.7mg/mL.Itis
outside the valid range of a clinical hemoglobinometer.
acceptable to expand the range to 0.02 to 0.8 mg/mL. The
8.4.1 Add 20 µL of well-mixed pooled whole blood speci-
cyanmethemoglobin reagent diluent serves as a zero blank in
men to 5.0 mL of cyanmethemoglobin solution or validated
thespectrophotometer.Measuretheabsorbanceat540nm.Plot
diluent.
a calibration curve from these values using hemoglobin con-
8.4.2 Allow the resulting solution to stand 15 min for
centration (mg/mL) on the y-axis and A on the x-axis. The
540 Drabkin’s or 5 min for cyanmethemoglobin reagent and then
calibration coefficient (F) is the slope of this plot. The
readtheabsorbanceofthesolutionwit
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: F756 − 13 F756 − 17
Standard Practice for
Assessment of Hemolytic Properties of Materials
This standard is issued under the fixed designation F756; 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 (´) indicates an editorial change since the last revision or reapproval.
1. 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. It is recommended that both tests (extract and direct contact) be performed unless the material
application or contact time justifies the exclusion of one of the tests.
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. Test Method E2524 provides a
protocol using reduced test volumes to assess the hemolytic properties of blood-contacting nanoparticulate materials; this may
include nanoparticles that become unbound from material surfaces.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 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.
1.7 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
E691 Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
E2524 Test Method for Analysis of Hemolytic Properties of Nanoparticles
F619 Practice for Extraction of Medical Plastics
F748 Practice for Selecting Generic Biological Test Methods for Materials and Devices
3. Terminology
3.1 Definitions:Definitions of Terms Specific to This Standard:
3.1.1 plasma hemoglobin—amount of hemoglobin in the plasma.
3.1.2 % hemolysis—free plasma hemoglobin concentration (mg/mL) divided by the total hemoglobin concentration (mg/mL)
present multiplied by 100. This is synonymous with hemolytic index.
3.1.3 comparative hemolysis—comparison of the hemolytic index produced by a test material with that produced by a standard
reference material such as polyethylene under the same test conditions.
3.1.4 direct contact test—test for hemolysis performed with the test material in direct contact with the blood.
This practice is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devicesand is the direct responsibility of Subcommittee F04.16
on Biocompatibility Test Methods.
Current edition approved Dec. 1, 2013March 1, 2017. Published January 2014April 2017. Originally approved in 1982. Last previous edition approved in 20082013 as
F756 – 08.F756 – 13. DOI: 10.1520/F0756-13.10.1520/F0756-17.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F756 − 17
3.1.5 extract test—test for hemolysis performed with an isotonic extract of the test material, material in contact with blood, as
described in Practice F619, in contact with the blood.
3.1.6 hemolysis—destruction of erythrocytes resulting in the liberation of hemoglobin into the plasma or suspension medium.
3.1.7 negative control—material, such as polyethylene, that produces little or no hemolysis (<2 % after subtraction of the blank)
in the test procedure. It is desirable that the control specimens have the same configuration as the test samples.
3.1.8 positive control—materials capable of consistently producing a hemolytic index (above the negative control) of at least
5 % (see 10.3). Although positive control materials have not been validated for this practice, washed Buna N rubber (Aero Rubber
Company; ARC-45010, 0.031 in. thick sheet) and vinyl plastisol (Plasti-Coat; 0.025 to 0.075 in. thick sheet, color:
DB1541-medium blue 300) produced hemolysis levels above 90 % when using extracts obtained at 121°C for 1 h during limited
interlaboratory round robin evaluations. In direct contact testing, Buna N rubber (ARC-45010) produced hemolysis levels of 14.5
6 5.3 %.
NOTE 1—The specific materials tested during the revision of this practice are available from Aero Rubber Company or Plasti-Coat. However, the
materials are not certified for this application, their shelf-life shelf life as positive controls has not been determined, and precision per Practice E691 has
not been established. Hence, all available materials may not be suitable as positive control materials for this application. Materials considered for use in
this application shall be checked for suitability in accordance with the requirements in this section. If you are aware of positive control materials, please
provide this information to ASTM International Headquarters.
3.1.9 cyanmethemoglobin reagent—reagent to which is added whole blood, plasma, or test supernatant that quickly converts
most of the forms of hemoglobin to the single cyanmethemoglobin form for quantification at its 540 nm spectrophotometric peak.
The reagent (based on that by van Kampen and Zijlstra, pH 7.0-7.4), is made with 0.14 g potassium phosphate, 0.05 g potassium
cyanide, 0.2 g potassium ferricyanide, and 0.5 to 1 mL of nonionic detergent diluted to 1 L with distilled water. The conversion
time of this reagent is 3 to 5 min. This reagent is recommended by the National Commission for Clinical Laboratory Studies
(NCCLS) and may be made from the chemicals or purchased from supply houses.
Aero Rubber Company, 8100 W. 185th St., Tinley Park, IL 60487, http://www.aerorubber.com; Plasti-Coat, 137 Brookside Dr., Waterbury, CT 06708, http://www.plasti-
coat.com. See Note 1.
Malczewski, R, Jackson, A, Lee, M, Malinauskas, R, Merritt, K, Peterson, L., “Standardizing an in vitro Hemolysis Assay for Screening Materials Used in Medical
Devices ,” Society for Biomaterials, Tampa, FL, Apr. 2002 (Extended abstract).
International Committee for Standardization in Haematology. J Clin. Pathol, Vol 49, 1996, pp. 271–274.
3.1.9.1 Discussion—
The first cyanmethemoglobin reagent used to measure total blood hemoglobin concentration was Drabkin’s reagent (1 g of sodium
bicarbonate, 0.05 g of potassium cyanide, 0.2 g of potassium ferricyanide and diluted with distilled water to 1 L). The
disadvantages of using the Drabkin’s reagent compared to the NCCLS cyanmethemoglobin reagent are that it has a conversion
time of 15 min and pH of 8.6, which may cause turbidity. However, Drabkin’s reagent is still available from commercial suppliers.
3.1.9.2 Discussion—
The Drabkin’s and cyanmethemoglobin reagents were developed to quantify the high hemoglobin concentration normally found
in whole blood (for example, 15 000 mg/dL). By modifying the sample dilution volumes and accounting for background
interference, these reagents can also be used to measure much lower plasma or supernatant hemoglobin concentrations as well
6,7
(Moore et al, Malinauskas).
3.1.10 PBS—phosphate buffered saline (Ca- and Mg-free). The use of phosphate buffered saline is preferable to the use of saline
in order to maintain the pH. The use of magnesium- and calcium-free PBS is necessary to maintain the anticoagulant properties
of the chelating agents used in collecting the blood. It is used as the background or “blank” for a hemolysis test.
x
3.1.11 A — absorbance value of cyanmethemoglobin reaction product measured at 540 nm, where “x” represents the specimen
in subsections 3.1.13 – 3.1.17.
3.1.12 F—slope of the hemoglobin standard curve. The units are [(mg/mL)/A] such that multiplication by an absorbance value
yields a hemoglobin concentration. Implicit assumption: The y-intercept of the hemoglobin calibration curve is approximately zero
and its effect on converting absorbance values to concentration values is negligible.
3.1.13 PFH—plasma free hemoglobin concentration.
3.1.14 C—total blood hemoglobin concentration.
Moore, G. L., Ledford, M. E., Merydith, A., “A micromodification of the Drabkin hemoglobin assay for measuring plasma hemoglobin in the range of 5 to 2000 mg/dl,”
Biochem. Med, Vol 26, 1981 , pp. 167–173.
Malinauskas, R. A., “Plasma hemoglobin measurement techniques for the in vitro evaluation of blood damage caused by medical devices,” Artificial Organs, Vol 21,
1997, pp. 1255–1267.
F756 − 17
3.1.15 T—diluted blood hemoglobin concentration.
3.1.16 B—blank (that is, no material added to this tube, only the isotonic medium).
3.1.17 S—sample (that is, test material sample, or negative and positive control sample).
4. Summary of Practice
4.1 Test and control material specimens or extracts are exposed to contact with rabbit blood under defined static conditions and
the increase in released hemoglobin is measured. Comparisons are made with the control and test specimens tested under identical
conditions. It is recommended that both tests (extract and direct contact) be performed unless the material application or contact
time justifies the exclusion of one of the tests.
5. Significance and Use
5.1 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.
5.2 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 F748.
6. Preparation of Test and Control Specimens
6.1 Samples should be prepared in accordance with Practice F619. A minimum total of six positive and six negative controls,
along with six test samples, should be prepared to be used in the direct contact test and the test with the extract (three samples
per test).
6.2 The final sample should be prepared with a surface finish consistent with its end-use application.
6.3 The sample shall be sterilized by the method to be employed for the final product.
6.4 Care should be taken that the specimens do not become contaminated during preparation but aseptic technique is not
required.
7. Hemoglobin Determination (Direct Method)
7.1 To create a hemoglobin concentration calibration curve using the cyanmethemoglobin method, use commercially available
reference standards and reagents from clinical diagnostic companies whichthat conform to the specifications of the International
5 8
Committee for Standardization in Hematology (ICSH). One commercial source is made by Pointe Scientific. A spectrophotom-
eter that provides absorbance readings to at least three decimal places places, and is able to detect the entire hemoglobin
concentration range (as specified in 7.2) should be used.
7.2 Prepare a standard curve from a suitable standard in six dilutions to accommodate the range of 0.03 to 0.7 mg/mL. It is
acceptable to expand the range to 0.02 to 0.8 mg/mL. The cyanmethemoglobin reagent diluent serves as a zero blank in the
spectrophotometer. Measure the absorbance at 540 nm. Plot a calibration curve from these values using hemoglobin concentration
(mg/mL) on the y-axis and A on the x-axis. The calibration coefficient (F) is the slope of this plot. The y-intercept should be
approximately zero.
NOTE 2—If local restrictions or other problems contraindicate use of these cyanmethemoglobin reagents, then another method for measuring total blood
hemoglobin concentration, plasma free hemoglobin concentration, and supernatant hemoglobin concentration may be substituted provided that it is
validated and shown to be substantially equivalent to the cyanmethemoglobin method. Methods which quantify oxyhemoglobin alone may not be
appropriate since some materials can convert oxyhemoglobin to other forms or alter the absorbance spectrum. Investigators should be aware that their
results of determining supernatant hemoglobin concentration may be compromised by absorption of hemoglobin by the test materials, precipitation of
hemoglobin out of solution, or alteration of the spectrophotometric absorbance spectrum by material leachables.
8. Collection and Preparation of Blood Substrates
8.1 Obtain anti-coagulated rabbit blood from at least three donors for each test day. The preferred anticoagulant is citrate (0.13
M). Approximately 5 mL should be drawn from each rabbit. Store the blood at 4 6 2°C and preferably use within 48 h. Blood
may be used up to 96 h after collection if the plasma free hemoglobin is not excessive. Equal quantities of blood from each rabbit
should be pooled.
8.2 Do not wash cells; use them suspended in th
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