ASTM F2382-24 循环血液接触医疗器械材料对部分凝血活酶时间（PTT）的评估

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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:F2382-24

INTERNATIONAL

Standard Test Method for

Assessment of Circulating Blood-Contacting Medical Device

Materials on Partial Thromboplastin Time(PTT)¹

This standard is issued under the fixed designation F2382;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(ε)indicates an editorial change since the last revision or reapproval.

1.Scope

1.1 This test method covers the screening of circulating

blood-contacting device materials for their ability to induce

blood coagulation via the intrinsic coagulation pathway.This

assay should be part of the hemocompatibility evaluation for

devices and materials contacting human blood,as per ANSI/

AAMIISO 10993-4.See also Practice F2888.

1.2 All safety policies and practices shall be observed

during the performance of this test method.

1.3 All plasma and any materials that had contact with

plasma will be bagged in a biohazard bag,properly labeled

with the contents,and disposed of by appropriate means.The

plasma should be handled at the Biosafety Level 2(BSL-2)as

recommended in the Centers for Disease Control(CDC)

National Institutes of Health(NIH)Manual Biosafety in

Microbiological and Biomedical Laboratories(BMBL,current

edition).

1.4 The normal pooled human plasma must have tested

negative for Hepatitis B(HBV)and Human Immunodeficiency

(HIV)viruses.The plasmas should be treated like any patient

plasma using standard precautions.The plasma should be

handled at the BSL-2 as recommended in the CDC/NIH

Manual BMBL.

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 appro-

priate safety,health,and environmental practices and deter-

mine the applicability of regulatory limitations prior to use.

1.7 This international standard was developed in accor-

dance with internationally recognized principles on standard-

ization established in the Decision on Principles for the

IThis test method is under the jurisdiction of ASTM Committe F04 on Medical

and Surgical Materials and Devices and is the direct responsibility of Subcommittee

F04.16 on Biocompatibility Test Methods.

Current edition approved Aug.15,2024.Published August 2024.Originally

approved in 2004.Last previous edition approved n 2018 as F2382-18.DOI:

10.1520/F2382-24.

Development of International Standards,Guides and Recom-

mendations issued by the World Trade Organization Technical

Barriers to Trade(TBT)Committee.

2.Referenced Documents

2.1 ASTM Standards:²

F2888 Practice for Platelet Leukocyte Count—An In-Vitro

Measure for Hemocompatibility Assessment of Cardio-

vascular Materials

2.2 ANSI/AAMI Standard:3

ANSI/AAMI/ISO 10993-4 Biological Evaluation of Medical

Devices—Part 4:Selection of Tests for Interactions with

Blood

2.3 Other Document:

U.S.Department of Health and Human Services Biosafety in

Microbiological and Biomedical Laboratories(BMBL),

5th ed.,1999⁴

3.Terminology

3.1 Definitions of Terms Specific to This Standard:

3.1.1 activator—a medical material which demonstrates a

shortened clotting time;an initiator of the intrinsic coagulation

pathway.

3.1.2 blank time—a period at the beginning of an assay

when no data is taken.This is done to eliminate interference

from premixing reagents,bubbles,and so forth.

3.1.3 duplicate flag—the agreement between the results of

duplicate samples in percent.For example,if set to "15,"the

difference between the two channels must be less than or equal

to 15%.If the variance in clot times exceeds this percentage,

an asterisk“*”will be printed by the average results on the

report.

²For referenced ASTM standards,visit the ASTM website,www.astm.org,or

contact ASTM Customer Service at www.astm.org/contact.For Annual Book of

ASTM Standards volume information,refer to the standard's Document Summary

page on the ASTM website.

³Available from American National Standards Institute(ANSI),25W.43rd St.,

4th Floor,New York,NY 10036,http://www.ansi.org.

4The BMBL5th Edition (December 2009)is available from the Government

Printing Office or https://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf

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F2382-24

3.1.4 equilibration time—the time allowed for the plasma

samples to warm to 37℃.The coagulation analyzer can be set

to zero if samples are pre-warmed to this temperature.

3.1.5 partial thromboplastin time(PTT)assay—a modifica-

tion of the Activated Partial Thromboplastin Time(APTT)

assay;unlike the APTT test,the PTT assay uses a reagent

(rabbit brain cephalin)without activating substances such as

silica,kaolin,elagic acid.The material being tested acts as the

activator.

3.1.6 read time—the time during which data is collected to

detect a clot.

4.Significance and Use

4.1 The purpose of this test method is to determine the time

citrated plasma exposed to medical materials takes to form a

clot when exposed to a suspension of phospholipid particles

and calcium chloride.In this test method,the test article is the

activator.The PTT assay is a general screening test for a

medical material's ability to activate the intrinsic coagulation

pathway.Material samples that show a shortened PTT are

activators of the intrinsic coagulation pathway.

4.2 The test article,reference materials,and controls are

exposed to human plasma.The plasma is tested on a coagula-

tion device.Each sample tube is assayed in duplicate.The

results are reported as a percentage of the negative control.

5.Apparatus

5.1 Polypropylene Test Tubes with Caps,12 by 75 mm.

5.2 Automatic Pipets and Tips,100 and 1000 μL.

5.3 Ice Bath.

5.4 Coagulation Analyzer;qualified,see A1.1.

5.5 Agitating Water Bath,37±2℃,capable of 60 rpm.

5.6 Coagulation Analyzer Cuvettes,or equivalent for spe-

cific analyzer.

6.Reagents and Materials

6.1 Calcium Chloride,25 mM.

6.2 Citrated Human Blood Plasma,fresh (less than 4h from

draw)or freshly frozen,maintained at minus 80℃,pooled

from at least three donors.

7.Hazards

7.1 The human blood plasma should be treated like any

patient plasma using standard precautions.The plasma should

be handled as recommended for BSL-2 by the CDC/NIH

publication Biosafety in Microbiological and Biomedical

Laboratories(BMBL,current edition).

8.Preparation of Apparatus

8.1 Prepare each test article,the negative reference

materials,marketed comparator device (if used),and controls

in triplicate.If a positive reference control material is used,a

single replicate is acceptable.All samples are prepared based

on a ratio of either 4 or,preferably 6 cm²of material to 1 mL

plasma and placed into polypropylene tubes.For device

testing,if test sample quantity allows,use three separate

devices;otherwise,take three representative samples from one

device.

8.2 Label duplicate polypropylene tubes and place in the ice

bath.

8.3 Turn on the coagulation analyzer and allow it to warm

up to 37±2℃ and equilibrate for at least 10 min.

8.4 Program the analyzer to test under the APTT function

with an equilibration time of 60 s,activation time of 120 s,a

read time of at least 300 s (that is,long enough to see plasma

clot formation),and an analyzer-specific blank time (if appli-

cable).

8.5 Pre-warm analyzer cuvettes(or cups,depending on

analyzer selected)at 37±2℃.

8.6 Pre-warm calcium chloride at 37±2℃.

8.7 Rabbit Brain Cephalin(RBC)Preparation:

8.7.1 Allow the RBC to come to room temperature.

8.7.2 Reconstitute the RBC as indicated by the RBC reagent

manufacturer.

8.7.3 Place in an agitating water bath set at 37±2℃ and

60 rpm for 15 min to ensure complete rehydration of contents.

8.7.4 Vortex 15 s after rehydration is complete.

8.7.5 Place at 37±2℃.

8.8 If using frozen blood plasma,quick thaw the plasma at

37±2℃ and place on ice immediately.

6.3 Lyophilized Rabbit Brain Cephalin(RBC).

6.4 Positive Reference Material(Optional),see A1.2.

6.5 Positive Control,glass(Pasteur pipette tips or glass

beads).Other qualified positive control materials such as

Buna-N-Rubber may be selected once they have demonstrated

a consistent thrombogenic response.

6.6 Negative Reference Material (for example,high-density

polyethylene,HDPE).

6.7 Marketed Comparator Device(Optional)—A legally

marketed,clinically acceptable device that has similar blood

contact nature and clinical use as the material/device being

investigated.

NoTE 1—It may be helpful to use a positive reference control material

(n=1)per assay to ensure continuity between runs.

9.Procedure

9.1 The test material(s),reference material(s),marketed

comparator device (if used),and controls are placed into

polypropylene tubes and exposed to the appropriate quantity of

plasma,based on a ratio of 4 cm²,or preferably 6 cm²of

material to 1 mL plasma.The negative control is a polypro-

pylene tube with 1 mL of plasma,without additional material.

9.2 The samples are exposed to the plasma for 15±1 min

in a 37±2℃ agitating water bath at 60 rpm.

9.3 After 15 min of incubation,the tubes are immediately

placed into the ice bath and immediately transferred into

pre-chilled new polypropylene tubes.

9.4 Vortex each sample 15 s before each use/run.

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9.5 Avoiding bubbles,transfer 100 μL of the plasma into

pre-warmed cuvettes and allow the plasma to equilibrate for

60s at 37±2℃.

9.6 To each cuvette/cup,add 100 μL warmed RBC

preparation,initiating the 2 min activation step.(Invert RBC to

mix prior to each use.)

9.7 After activation,add 100 μL warmed 25 mM calcium

chloride to each cuvette.

9.8 Allow the analyzer to read the sample for the formation

of clots(up to 5 min).

9.9 Record the clotting time(seconds)for each sample,as

well as the average clotting time of the duplicate samples.

NoTE 2—The volume ofplasma,RBC reagent,and calcium chloride

needed for the clotting time measurement may vary with different

anticoagulation analyzers.It is acceptable to use a plasma volume that is

specific to the coagulation analyzer used,as long as the plasma to reagent

ratio remains 1:1.

10.Calculation of Results

10.1 For each replicate,calculate the average clotting time

(from two readings)of the test sample,negative reference

control material,positive control material,and marketed com-

parator device,normalized to the negative control (untreated

plasma)values.In each case,the average clotting time is

expressed as a percentage relative to the negative control.

%negative control= (1)

10.2 Statistical Analysis of Results:

10.2.1 Calculate the mean values and standard deviations

for the test sample,negative control reference material,posi-

tive control material,and marketed comparator device by

averaging the respective percentage values from all three

replicates.

10.2.2 The positive control and the positive reference ma-

terial control (if used)results,expressed as percent negative

control(untreated plasma),must generally be<50%.However,

positive control results between 50 to 60%that of the negative

control may also be acceptable if the difference between the

negative reference control and the positive control is at least

30%(for example,if the result for the positive control=55%,

then the value for the negative reference control should be

>85%).The negative reference material control result,ex-

pressed as percent negative control(untreated plasma),must be

>80 %.If the assay does not meet this specification,the

experiment is to be repeated until the controls meet these

specifications.

10.2.3 The coefficient of variation(CV)between the dupli-

cates for each sample must be ≤15%.The duplicates of each

test article sample are averaged,and one value is reported as

the clotting time.This results in three clotting time values for

each test article.The three values are then averaged to report a

final average clotting time of the test article.The values for

each test article sample must be within±25%of this average.

If the values are greater than 25%of the average of the run,the

experiment needs to be repeated.

10.2.4 The values for the test article,negative control,

positive control,negative and/or positive reference material,

marketed comparator device results are compared to each other

with analysis of variance(ANOVA)including post-hoc pair-

wise comparisons such as a Tukey or Newman-Keuls test.

10.2.5 Differences shall be considered statistically signifi-

cant if the p-value is less than 0.05.The test article is

considered to pass the PTT test if there is no statistical

difference between the test article and the negative control

(untreated plasma)or the negative reference material control.

11.Assay Validity

11.1 The positive control and the positive reference material

control(if used)results,expressed as percent negative control

(untreated plasma),must generally be <50 %.However,posi-

tive control results between 50 and 60%of that of the negative

control may also be acceptable if the difference between the

negative and the positive control is at least 30%(for example,

if the result for the positive control=55%,then the value for

the negative reference control should be >85%).The negative

reference material control result,expressed as a percent nega-

tive control (untreated plasma),must be >80%.If the assay

does not meet this specification,this assay is to be repeated

until the controls meet these specifications.

11.2 The coefficient of variation(CV)between the dupli-

cates for each sample must be ≤15%.The duplicates of each

test article sample or legally marketed comparator device (if

used)sample are averaged and one value is reported as the

clotting time.This results in three clotting time values for each

test article and legally marketed comparator device (if used).

The three values are then averaged to report a final average

clotting time of the test article or legally marketed comparator

(if used).The values for each test article sample must be within

±25%of this average.If the values are greater than 25%of

the average of the run,the experiment needs to be repeated.

12.Test Evaluation

12.1 The test article is also considered to pass the PTT test

if the test article results are>80%(acceptance criterion for the

negative reference material)and the test article is at least 30%

above that of the positive control.If the results of the test

article are≤50%(acceptance criterion for a positive control),

then the test article is considered to fail this screening test.

12.2 If the test article results fall between these assay

acceptance criteria of 50 to 80%of the negative control,it is

recommended that the results from the test article be compared

to results from a legally marketed comparator device(that is,in

a repeat test with all controls).If the results for the test article

are less than that of a comparator,then additional thromboge-

nicity evaluation is recommended(for example,risk

assessment,animal study,or other thrombogenicity tests,or a

combination thereof,on the finished device under more clini-

cally relevant conditions)

13.Precision and Bias

13.1 The precision and bias determination of this test

method is not relevant because this is a biological assay(not an

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ThisinternationalstandardwasdevelopedinaccordancewithinternationallyrecognizedprinciplesonstandardizationestablishedintheDecisiononPrinciplesfortheDevelopmentofInternationalStandards,GuidesandRecommendationsissuedbytheWorldTradeOrganizationTechnicalBarrierstoTrade(TBT)Committee.Designation:F2382-24I...

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ASTM F2382-24 循环血液接触医疗器械材料对部分凝血活酶时间（PTT）的评估

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