Tiêu chuẩn iso 10993 4 2017

The a eutic a he esis eq ipment an devic s for adsorption of spe ific subs anc s from the blo d, be a use of their high surfac -t o-v lume ratio, can pot entialy activ t e complement, co

Biolog ical evaluation ofmedical

Part 4:

Selection oftests for inter actions

w ith blood

Év lu tion biolo ique des dispositifs médica x —

Partie 4 : Ch ix des e ssais p ur le s inte ac tions a e le san

T ir d edition

2 17-04

Reference n mb r

ISO 1 9 3-4:2 17(E)

COPYRIGHT PROTECTED DOCUMENT

© ISO 2017, P blshed in Sw itz rlan

A ll rig hts r eserved Unles otherw ise spe ified, nopar of this p blc tion ma y be r epr od c d or utilz d otherw ise in an form

or b an me ns, ele tr onic or me hanic l, inclu in p oto opying , or postin on the internet or an intranet , w ithout prior

written permis ion Permis ion c n be req esed from either ISO at the ad r es below or ISO’s member bod y in the c u try of

F reword i v

Introduction vi

1 Sc ope 1

2 Nor mati ve r eferenc es 1

3 Terms an definitio s 1

4 A bbreviated terms 4

5 T ypes of devic es in c ontact w ith blo d (as categ orized in ISO 10993-1) 5

5.1 Non-blo d-contact devic s 5

5.2 Ex te nal commu icating devic s 5

5.2.1 General 5

5.2.2 Ex te nal commu icating devic s that e ve as an in ir ect blo d p th 5

5.2.3 Ex te nal commu icating devic s dir ectly contacting cir culating blo d 5

5.3 Implant devic s 6

6 Character izatio of blo d interactio s 6

6.1 General r eq ir ements 6

6.2 Categ ories of tests an blo d inter actions 1

6.2.1 Re ommended tes s for interactions of devic s with blo d 1

6.2.2 Non-contact devic s 1

6.2.3 Ex te nal commu icating devic s an implant devic s 1

6.2.4 Limitations 1

6.3 T ypes of tests 1

6.3.1 In vitro tes s 1

6.3.2 Ex viv tes s 14

6.3.3 In vivo tests 14

A nne x A (informative)Preclnical evaluatio of cardio as ular devic es and prostheses 16

A nne x B (informative) Rec ommended lab rator y tests — Pr inciples, s ientific basis and inter pr etatio 21 A nne x C (informative) Thr omb sis — Metho s for in vivo testing 32 A nne x D (informative) Ha matolog y/ha mol ysis — Metho s for testing — E aluation of ha mol ytic proper ties of medical devic es and medical devic e mater ials 39 A nne x E (informative) C mplement — Metho s for testing 46

A nne x F (informative) Les c ommon laborator y tests 49

A nne x G (informative) Tests which are not rec ommended 53

Biblog raphy 55

ISO (he Int ernational Org nization for Stan ardization) is a worldwidefede ation of national s an ards

b dies (ISO membe b dies) The work of pr p ring Int ernational Stan ards is normaly car ied out

through ISO t ech ical committ ees Each membe b dy int er st ed in a subje t for w hich a t ech ical

committ ee has be n es a lshed has the right t o be r pr sent ed on that committ ee Int ernational

org nizations, g ove nmental an non-g ove nmental, in laison with ISO, also take part in the work

ISO cola orat es closely with the Int ernational Ele trot ech ical C mmis ion (IEC) on al matt ers of

ele trot ech ical s an ardization

The proc d r s used t o develo this document an those int en ed for it furthe maint enanc ar

desc ibed in the ISO/IEC Dir ctives, Part 1 In p rticular the dife ent a pro al c it eria ne ded for the

dife ent ty es of ISO document should be not ed This document was draft ed in ac ordanc with the

edit orial rules of the ISO/IEC Dir ctives, Part 2 ( e www iso org dir ctives)

A tt ention is drawn t o the p s ibi ity that some of the element of this document ma be the subje t of

p t ent right ISO shal not be held r sponsible for identifying any or al such p t ent right Detais of

any p t ent right identified d ring the develo ment of the document wi be in the Introd ction an / r

on the ISO ls of p t ent de larations r c ived ( e www iso org p t ent )

Any trade name used in this document is information given for the convenienc of use s an does not

cons itut e an en orsement

F or an ex lanation on the v lu tary natur of s an ards, the meaning of ISO spe ific t erms an

ex r s ions r lat ed t o conformity as es ment, as wel as information a out ISO’s adhe enc t o the

World Trade Org nization (WTO) principles in the Te hnical Bar ie s t o Trade (TBT) se the folowing

URL: www iso org iso/ for word html

This document was pr par d b Te h ical Committ ee ISO/TC 1 4, B iolo ic al a d clinic al ev lu tio o

medic al dev ic es

This third edition canc ls an r plac s the se on edition (ISO 1 99 -4:2 0 ), w hich has be n

t ech icaly r vised

It also incorporat es the Amen ment ISO 1 99 -4:2 0 /Amd 1:2 0

The folowing chang es wer made:

a) some definitions ha e be n r vised an new def initions ha e be n ad ed;

b) Ta les 1 an 2 ha e be n consoldat ed int o a single new Ta le 1 with t es cat eg ories an heade s

r org niz d t o emphasiz an inclu e mat erial an me hanical-in uc d haemoly sis t es ing an in

v itro an in v i vo t es ing for as es ment ofrisk for thromb sis;

c) Ta les 3 an 4 ha e be n consoldat ed int o a single new Ta le 2 with a simplf ied l s of sugg est ed

an mos common t es s;

d) Annex B has be n updat ed t o co e only the mos common practic d t es s for as es ing blo d

int eractions;

e) Annex Chas be n ad ed t o co e the t opic of in v i vo thromb sis an methods for t es ing;

f ) Annex D, w hich was Annex C in the pr vious edition, has be n updat ed an now inclu es ad ed

information on me hanicaly-in uc d haemoly sis;

g) Annex E has be n ad ed t o co e the t opic of complement t es ing an bes t es method practic s;

h) Annex es F an G have be n ad ed t o pr sent the les common t es s used t o as es int eractions with

blo d and the t es s that ar not r commen ed for pr clnical as es ment of medical devic blo d

int eraction, r spe tively Many of these methodswe e pr viously inclu ed in Annex B;

i) subtle languag e r finement can be fou d throughout the r vised documen

j) theBibl o ra hy has be n r org niz d b common subje t of int er s an updat ed with ad itional

an mor cur ent r fe enc s

The sele tion an design of t es methods for the int eractions of medical devic s with blo d should take

int o conside ation devic design, mat erials, cl nical uti ity, usag e environment and risk benef it This

level of spe ificity can only be co e ed in ve tical s an ards

The initial sour e for develo ing this document was the publcation, Guidelnes fr blo d mate ial

inte ac tions , R ep rt of the National Heart, Lung, an Blo d Ins itut e

[14]

cha t ers 9 an 1 This

publcation wassubseq ently r vised

[1 ]

Biolog ical evaluation ofmedical devic es —

a) a clas if ication of medical devic s that ar int ended for use in contact with blo d, b sed on the

int en ed use an d ration of contact as defined in ISO 1 9 3-1,

b) thefu damental principles g ove ning the ev luation of the int eraction of devic s with blo d,

c) the rationale for s ructur d sele tion of t es s ac ording t o spe if ic cat eg ories, t og ethe with the

principles an scientif ic basis of these t es s

Detaied r q ir ment for t es ing cannot be spe ified be a use of lmitations in the k ow ledg e an

pr cision of t es s for ev luating int eractions of devic s with blo d This document desc ibes biolo ical

ev luation in g ene al t erms an ma not ne es ari y pro ide suff icient guidanc for t es methods for a

spe if ic devic

The chang es in this document do not in icat e that t es ing con uct ed ac ording t o prior ve sions of

this document is inv l d F or market ed devic s with a hist ory of safe cl nical use, ad itional t es ing

ac ording t o this r vision is not r commen ed

2 Normati ve r eferences

The folowing document ar r fe r d t o in the t ext in such a wa that some or al of their cont ent

cons itut es r q ir ment of this document F or dat ed r fe enc s, only the edition cit ed a ples F or

un at ed r fe enc s, the lat es edition of the r fe enc d document ( inclu ing any amen ment ) a ples

ISO 1 9 3-1, B iolo ic al ev lu tio o medic al dev ic es — Part 1: Ev lu tio a d tes tin w ithin a ri sk

ma a ement proc es s

ISO 1 9 3-1 , B iolo ic al ev lu tion o medic al dev ic es — Part 12 : S mple pr p ratio a d r fe enc e

mate ial s

3 Terms and definitions

F or the purposes of this document, the t erms an definitions given in ISO 1 9 3-1, ISO 1 9 3-1 an

the folowing a ply

ISO an IEC maintain t erminolo ical data ases for use in s an ardization at the folowing ad r s es:

— IECEle tro edia: a aia le at ht p:/ www ele tro edia org

— ISO Onlne browsing plat orm:a ai a le at ht p:/ www iso org o p

anticoa ulant

ag ent w hich pr vent or delay s blo d co gulation

E AMP E Heparin, ethy lenediaminetetra cetic acid (EDT A), sodium citrate

3.2

blo d/device interactio

int eraction betwe n blo d or a blo d comp nent and a devic

3.3

coa ulation

phenomenon that r sult from activ tion of the clot ing (co gulation)fact or cascade

Not e 1to entry: F actor of the co gulation cas ade an f ibrinolytic systems can b me sured folowing e posure

t o devices either in v itr or in v i vo

3.4

complement sy stem

p rt ofthe innat e immune syst em consis ing of o e 3 dis inct plasma prot eins, inclu ing enzymes,

cofact ors, an c lular r c pt ors w hich ma be involved in the promotion of thromb sis

Not e 1 t o entry: Efector molecules prod ced from complement components are pos ible components in the

p enomena of inflammation, p a ocyt osis an cel ly sis Complement activation related to immu ot oxicity,

h per ensitivity an g neration of anap y lato ins is not covered in this document ( Se ISO /TR 1 9 3-2 )

Note 2 t o entry: T e focus in this document is complement activation as it can promote an ac elerat e ha molysis,

platelet an leukocyte activation an thromb sis on device mat erial surfaces ( Se also An e E on complement

activation.)

3.5

direct blo d co tact

t erm used w hen the devic or devic mat erial comes int o phy sical contact with blo d or blo d

cons ituent

3.6

emb lzatio

proc s w he eb a blo d thrombus, or for ign o je t, is car ied in the blo ds r am and w hich ma

be ome lodg ed an cause o s ruct ed blo d flow downs r am

3.7

e vivo test sy stem

t erm a pled t o a t es sy st em that h nt blo d dir ctly from a h man subje t or t es animal int o a t es

chambe locat ed out ide the b dy

Not e 1t o entry: If using an animal model, the blo d may b sh nted directly b ck into the animal (recir ulating)

or col ected in t est tub s for evaluation (single pass) In either case, the test chamb r is located outside the b d

ha mocompatible

<devic or devic mat erial> a le t o come int o contact with blo d without any a pr cia le cl nical

y-signif icant adve se r actions such as thromb sis, h emolys i s (3.1 ), plat elet, leukocyt e, an complement

activ tion, an / r othe blo d-as ociat ed adve se event oc ur ing

3.12

in irect bloo contact

natur of devic s that contact the p tient’ s blo d p th at one p int an se ve as a con uit for entry int o

the v scular syst em

E AMP E Drug an parent eral n trition solution delivery devices

3.13

le al y-marketed comparator device

LMCD

a pro ed, or clear d long -es a lshed, an r co niz d-t o-be-safe medical devic used as a r fe enc

control in an in vitro or in vi vo safety ev luation of a t es devic of simi ar design, mat erial(), an

clnical use

Note 1 to entry: It may b neces ary that the LMCD b le al y marketed in the same re ion as the re ulat ory

submis ion for the test device

3.1

no -bloo -co tact

natur of the devic or mat erial contact with the p tient’ s b dy w he e the devic or p t entialy

extract ed mat erial does not have dir ct or in ir ct contact with blo d

3.1

col oidal osmotic pres ure

total influenc of the prot eins or othe larg e mole ular mas subs anc s on the osmotic activity of plasma

3.1

platelets

an clear, c lular b dies that ar pr sent in blo d an contribut e t o the proc s of thromb sis b

adhe ing t o surfac s, r leasing fact ors, and/ r a gr g ting t o form a haemos atic plug

3.17

platelet adherent

<mat erial or devic > ha ing the t en ency t o alow or promot e platelet (3.1 )t o at ach t o it surfac

Note 1 t o entry: T is is of en characterized relative to a ne ative control, positive control, an / r LMCD u on

blo d contact d e t o its surface properties

Note 2 to entry: Platelet ad erent do s not neces arily me n platelet activating, i.e plat elets on a surface may or

may not b activated

thromboembol zation

proc s w he e a dislodg ed thromb s (3.2 ) is car ied downs r am, w he e it ma cause subseq ent

v scular blockag e or oc lusion

formation of a thromb s (3.2 ) u de in v i vo, ex v i vo, or in v itro simulat ed con itions, caused b

activ tion of the co gulation sy st em and platelets (3.1 )in flowing w hole blo d

Not e 1t o entry: T romb sis can also oc ur in re ions of a blo d ves el or device where there is stasis

3.23

w hole blo d

unfractionat ed blo d drawn from a h man donor or t es animal

Not e 1 to entry: T e blo d may b non-antico gulated or antico gulated, e.g contain sodium citrate or heparin

as an antico gulant

4 A bbreviated terms

Bb enzymaticaly active fra ment of Fact or B prod c d b clea ag e (b F act or D )in the

activ tion of the alt ernative p thwa

C4d degradation prod ct of C4 b clas ical p thwa complement activ tion

C a, C a complement splt prod ct from C an C

CH-5 amount of complement r q ir d t o lyse 5 % of a RBCsuspension

D-Dime spe ific f ibrin degradation prod ct (F XI Ic os -l nked fibrin)consis ing of

D-fra ment dime

ELISA enzyme-l nked immu osorbent as a

FDP fibrin/fibrinog en degradation prod ct

F 2 the non-catalytic fra ment splt of from prothrombin in it conve sion t o thrombin (also

r fe r d t o as F1 + 2)

iC b inactive form of C b, a sub-fra ment of C

IFU ins ruction for use

IVC infe ior vena ca a

P T p sitron emis ion t omo ra hy

PF-4 plat elet act or 4

PRP plat elet-rich plasma

PTT p rtial thromb plas in time

SC b-9 prod ct of t erminal p thwa complement activ tion

SEM scan ing ele tron mic osco y

TC t erminal complement complex; also caled membrane at ack complex (MAC); es imat ed

b measuring SC b-9

5 T ypes ofdevic es in c ontact with blood (as categ or ized in ISO 10993-1)

5.1 Non-blood-c ontact devic es

Non-blo d-contact devic s ar devic s that do not ha e dir ct or in ir ct contact with eithe blo d or

blo d cons ituent that r side in the b dy or that ar r turned t o the b dy An in v itro dia nos ic devic

an a blo d-col e tion tube ar ex mples of non-blo d-contact devic s Some devic s, such asintrod c r

sy st ems for implant , ma contain b th blo d-contacting an non-blo d-contacting comp nent

5.2 Ex ternal c ommunicating devic es

5.2.1 General

These ar devic s that contact the cir ulating blo d an se ve as a con uit int o the v scular syst em

Some devic s ma have component or p rtions with dife ent ty es of contact (dir ct and in ir ct)

Ex mples inclu e but ar not l mit ed t o the folowing

5.2.2 Ex ter nal c ommunicating devic es that serve as an indir ect blo d path

— blo d cole tion devic s;

— cann lae;

— c l sa e s;

— devic sfor the st orag e an adminis ration of blo d an blo d prod ct (e.g tubing an b gs);

— ext ension set ;

— intra ascular cathet ers

5.2.3 Ex ter nal c ommunicating devic es directl y c ontacting circulating blo d

— athe e t omy devic s;

— blo d monit oring devic s with dir ct or in ir ct blo d contact ;

— cardio ulmonary b pas cir uitry;

— devic sfor adsorption of spe if ic subs anc s from blo d;

— donor an the a eutic a he esiseq ipmen

— extracorp r al membrane o y g enat ors;

— haemodialy sis /haemofiltration devic s;

— int erventional cardiolo y an v scular devic s;

— intra ascular cathet ers (b lo n, ima ing, lase , ultrasou d);

— leu ocyt e r mo al f ilt ers;

— pe cutaneous cir ulat ory sup ort devic s;

— r tro rade coronary pe fusion cathet ers;

— v scular guidewir s

5.3 Implant devices

Implant devic s ar plac d larg ely or entir ly within the v scular sy st em Ex mples inclu e but ar not

l mit ed t o the folowing:

— an ulo las y rings;

— art erio enous sh nt ;

— blo d monit ors ( implanta le);

— cir ulat ory sup ort devic s (ventricular-as is devic s, artificial heart , intra-a rtic balo n pumps);

— emb lzation devic s;

— endo ascular synthetic v scular grafs;

— implanta le def ibri lat or an cardio e t er leads;

— infe ior vena ca a f ilt ers;

— int ernal drug delvery cathet ers;

— intra ascular o y g enat ors (artificial lu gs);

— me hanical or tis ue heart v lves;

IMPORTANT — Since this is a h rizo tal International Standard, so nd rationales can be

sup l ed to justify the ch ice of test categ ory (ies) based o the device being characterized F or

example, in vivo testing for evidence of thrombosis is frequently the prefer ed metho for device

characterizatio in the thromb sis categ ory However, in some cases, written rationales that

include a combinatio of tests from the categ ories of coa ulatio , platelets, ha matology and

complement can be used as a substitute for thrombosis testing

6.1.1 Fig ur e 1 i us r ates a de ision tr ee that can be used to dete mine whethe tes ing for interaction

with blo d is ne es ary Blo d inter actions can be divided into several categ ories b sed on the primary

pr oc s or sys em being measur ed Ta le 1 ls s examplesof devic s which contact cir culating blo d an

the categ ories of testing a pr opriate to each devic The ls is not al inclusive an sou d ju g ement

shal be a pled to devic s not ls ed in the tables

F or medical devic s w he e a spe ific Int ernational Stan ard (ve tical s an ard) exis s, the biolo ical

ev luation r q ir ment an t es methods set forth in that ve tical s an ard shal take pr c denc

o e the g ene al r q ir ment sugg est ed in this document

6.1.2 Wher e p s ible, tes s shal use an a propriate model or sys em which simulates the g eometry

and con itions of contact of the devic with blo d d ring clnical a plcations The simulation should

inclu e an a propriate d r ation of contact , temperatur e, s e ie condition, antico g ulant (an level; se

6.1.1 ) an flow con itions For example, for devic s of defined g eometry such as a v scular s ent , the

surfac area used in the test ,in cm

2

, shal be g iven consideration relative to the fluid v lume of the invitro

tes sys em For devic s w ith u defined or complcated g eometry ( uch as a dispe sion of PV particles

used as an emb lzation ag ent), mas should be used ins ead of surfac ar ea to dete mine the amou t of

sample used in tes sys em

Only dir ct or in ir ct blo d-contacting part should be t est ed The sele t ed t es methods an

paramet ers should be in ac ordanc with the cur ent s at e of the art

Ap ro riat e ty e an level of antico gulant ma be case spe if ic depen ing on both the devic

use in ication an the ty e of t es con uct ed Inclu e information on the spe ific ty e an level of

antico gulation used an pro idea discus ion on the a i ity t o disc rn p sitive an neg tive r sp nses

F or furthe information, se 6.1.6 an C.2 for animal s u ies, 6.1.1 for in vi vo an ex v ivo t es s, 6.3.1for

in v itro t es s an A.3for cathet ers an guide wir s

As many t es s for haemocompatibi ity ar r co niz d t o be s rictly surfac -contact depen ent, such

t es s (e.g complement activ tion) wi l not a ply t o in ir ct contact a plcations

6.1.3 C ntrols (p sitive and negative) shal be used u les their omis ion can be jus ified Wher e

p s ible, tes ing should include a relev nt pr edicate devic alr ead y in clnical use (i.e a LMCD) or a wel

-characte iz d mate ial

6]

Controls should inclu e neg tive an p sitive r fe enc mat erials Al mat erialsand LMCDs t est ed shal

me t al q alty control and q alty as uranc spe if ications of the man factur r an t es la orat ory

Al mat erials an devic s t est ed shal be identif ied as t o sour e, man factur r, gradean ty e

6.1.4 Testing of mate ials which ar e can idates for comp nents of a devic ma y be con ucted for

scr eening purp ses Howeve , such pr elminary tests do not e ve as a subs itute for ther eq ir ement that

the complete s e i z d devic or devic component should be tes ed u de con itions which simulateor

exag g erate clnical a plcation

NOTE 1 C ang s in man facturing proces (inclu ing use of man facturing aids)that could afect he surface

properties, or chemistry of the complet e st erilized device, could also impact ha mocompatibility

NOTE 2 Where a ing could impact the f inal device properties, use of a ed samples can also b neces ary (For

e ample, the properties of biolo ical y active co tings such as heparin could chang e over time.)

6.1.5 Tests which do not simulate the con itions of a devic d ring use ma y not pr edict ac ur ately

the natur e of the blo d/devic interactions which can oc ur d ring clnical a plcations In ad ition, the

ca acity of short-te m in vitro or e x viv tes s to pr edict pe formanc in actual clnical a plcations is

thought to be highe when the clnical a plcation inv lves lmited ex posur e r athe than pr olong ed or

pe manent ex osur e

NOTE Simplif ied t esting of can idate device materials (e.g surface g ometric an fu ctional chemical

modif ications) can serve as a c ucial step in device material identif ication,optimization an selection

6.1.6 If an animal s ud y is to be con ucted, devic s whose inten ed use is e x vivo (ex te nal

commu ication) should be tes ed e x vivo an devic s whose inten ed use is in vivo (implant ) should be

tes ed in viv in an animal model simulating as closely as p s ible con itions of clnical use Pr otocols in

such investigations should spe ificaly cal out each tes categ ory ( e 6.2.1) being ev luated an desc ibe

the spe ific method( ) of as es ment

a

F or dir ct an in ir ct contact devic s, thene es ity for haemocomp tibi ity t es ing should be

con-side ed b sed upon a pro riat e risk analy sis, inclu ing prior haemocomp tibi ty t es ing, cl nical data,

extracta le /leacha le data, an / r information on surfac charact eris ics F r ex mple, for devic s

with dir ct contact, extracta le /leacha le t es ing ma not be sufficient if the surfac morpholo y is

chang ed, even if the extracta le /leacha le chemis ry is the same ( e ISO 1 9 3-1)

Figure 1 — Decisio tre to help determine w hethe testing for interaction with blo d is

neces ary

c

Xc

Xc

Xc

c

Xc

Xc

Xc

b

c

Xc

c

Xc

b

c

Xc

c

Xc

c

Xc

c

Xc

c

Xc

6.1.7 In vitro tests ar e r eg ar ded as useful in scr eening exte nal commu icating dev ic s or implants

and p tential early inter actions between dev ic s/mate ials w ith blo d, but ma y not be ac ur ate

pr edictors of blo d/dev ic inter actions oc ur ing up n pr olong ed or r epeated ex osur e or pe manent

contact se 6.3.1)

NOTE F or new devices or devices where there is a chang in g ometry, testing u der p ysiolo ic flow can b

ne ded For long -term catheter or permanent implants, in v itr t est sy stems might not b suff icient d e t o blo d

sta ility is ues

6.1.8 Devic s or devic component which come into ve y brief/tr ansient contact with circulating

blo d (e.g lanc t , hypode mic ne dles, ca i ary tubes that are used for les than 1 min) g ener aly do

not r eq ir e blo d/devic interaction tes ing

NOTE 1 F or prod cts made with mat erials such as co tings that could b lef in contact with blo d af er the

device is removed, blo d/device interaction testing might b neces ary

NOTE 2 If some device components (e.g syring e b dies) are in contact with fluids that wil ultimately b

injected into the patient, an the st ora e time is u specif ied or gre ter than 1 min, ha molysis t esting of the

fluid-contacting component would b ne ded, even though the device itself would b in contact with cir ulating

blo d for les than 1 min

6.1.9 Disp sa le la oratory eq ipment used for the cole tion of blo d and pe formanc of in vitro

tests on blo d shal be ev luated to asc rtain that ther e is no sig nificant inte ferenc with the tes being

pe formed

6.1.10 If tests ar e sele ted in the man e desc ibed an testing is cond cted u de con itions which

simulate clnical a plcations, the r esult of such tes ing ha ve the g r eates prob bi ty of predicting

clnical pe formanc of devic s For devic s that o erate o e a rang e of con itions, the ex tr eme an the

a verag e con itions should be consider ed Howeve , spe ies diferenc s an othe factors ma y lmit the

pr edicta i ty of any test

6.1.11 Be ause of spe ies diferenc s in blo d r eactivity, human blo d should be used wher e pos ible

(with the exc ption of establshed tes methods w ith animal blo d, such as some haemolysis tes s)

When animal models are ne es ary, for example for ev luation of devic s used for pr olong ed or r epeated

ex posur e or pe manent contact ,spe iesdifer enc s in blo d reactivity shal be consider ed

Blo d v lues an r activity in h mans an non-h man primat es ar ve y simi ar

[2 4]

Theuse of animals,

such as the ra bit, pig, calf, she p or do , can also be ac epta le for a p rticular ty e of t es Howeve ,

sinc spe ies dife enc s ma be significant (for ex mple, plat elet ad esion

t end t o oc ur mor r adi y in the canine than in the h man), al r sult of animal

s u ies shal be int erpr t ed with caution The spe ies sele t ed an the n mbe of animals used shal be

jus if ied ( e also ISO 1 9 3- 2)

NOTE T e use of non-h man primat es for in v i vo blo d compatibility an medical device t esting is prohibited

by EU law ( 86 /609/E C)an some national laws

6.1.12 T e use of antico g ulant in in viv an e x viv tests should be a voided u les the devic is

desig ned to pe form in their pr esenc The type an conc ntration of antico g ulant used influenc

blo d/devic interactionsan their sele tion shal be jus ified Devic s that ar e used with antico g ulant

should be as es ed using antico g ulants in the rang e of conc ntr ations used clnicaly an / r desc ibed

in the prod ct IFU or othe a propriate lteratur e Spe ies diferenc s should also be consider ed when

dete mining the a propriate level of antico g ulation

6.1.13 Modifications in a clnicaly ac epted devic shal be considered for their efe t on blo d/devic

interactions an cl nical fu ctions Examples of such modifications inclu e chang es in desig n, g eometry,

chang es in surfac or bulk chemical comp sition of mate ials an chang es in tex tur e, p r osity or

othe prope ties A n in vitro flow model w ith a plcation-consis ent ex posur e con itions an r elev nt

measurement can be used to ev luate the efe t of modifications to a clnicaly ac epted devic

6.1.14 A suficient n mbe of r eplcations of a tes inclu ing suitable contr ols should be pe formed to

pe mit s atis ical ev luation of the data The v ria i ty in some tes methods r eq ir es that those tes s

be r epeated a suficient n mbe of times to dete mine sig nificanc In ad ition, r epeated studies o e an

exten ed pe iod of blo d/devic contact pr ovide infor mation a out the time-depen enc of the blo

d-devic interactions

[2 3]–[2 6]

Balanc should be consider ed betwe n s atis ical ev luation an animal

welfare when a plying in vivo tes ing ; se ISO 1 9 3-2

6.1.15 T e r ecommen ations within 6.1, tog ethe with Fig ure 1 and Ta le 1, se ve as a g uide for the

sele tion of tests ls ed in Ta le 2 Furthe g uidanc on pr e-clnical ev luations is g iven in An exes A to G

In summary, the folowing pr oc d r e shal be pe formed:

a) det ermine w hich p t ential blo d int eraction cat eg ories ( e 6.2) ar a pro riat e for conside ation

t o es a l sh safety ofthe p rticular devic ( e ex mples in Ta le 1);

b) ev luat e the ex i s tin in ormatio in each t es cat eg ory for the devic ;

c) w he e s uffic ient s afet y inormatio ex i s ts , pr p r an a pro riat e rationale t o sup ort this

conclusion an that furthe t es ing is not ne es ary;

NOT An diference in formulation, g ometry, surface properties, fa rication methods, sterilization

tech iq e an / r clinical use could limit he use of safety information on a similar prod ct

d) w he e ins uffic ient in ormatio ex i s ts un e a t es cat eg ory( ies), sele t a pro riat e t es s, b sed up n

ex mplesin Ta les 1 an 2, t o sup ly the ad itional safety information

6.2 Categ or ies of tests and blood interactions

6.2.1 Rec ommended tests for interactions of devic es with blo d

R ecommen ed t es s ar org niz d on the b sis of the ty e of devic ( e ex mples in Ta le 1) The t es s

ar divided int o the folowing cat eg oriesb sed on the primary proc s or sy st em being measur d:

Table 2 — Commo tests used to as es interactio with bloo

Tes s b cat eg ries

Haemoly sis Material-n uced (e.g ASTM

[1 ]

, NIH[2 ]

,

MHLW[2 ]

)

Mechanical-n uced

Thromb sis

(in v i vo, e v i vo)

Gros analy sis , per enta e oc lusion, light

mic os op , SEM

In vitro thromb sis

C a ulation T rombin (e.g TAT, F1.2), f ibrin (e.g F A)as ay s,

P T as ay

Plat elet activ tion Platelet cou t % loss) an some in icat or of

activation (e.g rele se prod cts or plat elets

surface marker such as β G, P 4, TxB2)or SEM

(platelet morp olo y)

Haemat olo y Complete blo d cou t C C), leucocyte activation

C mplement sy st em SC5 -9 (C3 optional)

a

Inclu ed in al animal s u ies ( e B.2.1 an ISO 10 93-6)

Not al tes s are ne ded for e ch c tegory an tes in in e ch c tegory mig t not be

eq ivalent

6.2.2 No -c ontact devic es

These devic sdo not r q ir blo d/devic int eraction t es ing

6.2.3 Ex ter nal c ommunicating devic es and implant devic es

Aft er using Ta le 1 t o algn a new devic u de inves ig tion with simi ar exis ing devic s an noting

the t es cat eg ories for conside ation, use Ta le 2, An ex es A an E t o guide the sele tion of a pro riat e

t es s for as es ing blo d int eractions

6.2.4 Limitatio s

Tes ing an s u y design p ramet ers ma pr sent c rtain practical lmitations /conside ations b sed

up n scienc , t ech olo y an the p rticular a plcation F or ex mple:

a) mat erials /devic s in a high blo d flow (art erial) environment ma int eract with blo d dife ently

in a low blo d flow (venous) environmen

b) blo d int eractions ma oc ur with al mat erials, i.e the t es mat erials /t es devic s a d the non-t es

mat erials (e.g t es sy st em) C ution shal be taken t o not confoun blo d int eractions as ociat ed

with the t es mat erials t o those contribut ed b othe fact ors;

c) s u ies that r ly on jus one ty e of t es for blo d int eractions ma be les pr dictive of the true

r sp nse than s u ies that inclu e seve al dife ent t es s for blo d int eractions;

d) immu o s ay s for det ecting prot ein in icat ors of haemocompatibi ity, e.g T AT, C a, et c ar oft en

a ai a le for h man blo d t es ing but ar not g ene aly a aia le for use or functional with blo d

from othe spe ies

6.3 T ypes of tests

6.3.1 In vitr tests

In v itro t es ing (models) should conside designs t o simulat e the anticip t ed wors -case cl nical use

con itions of each devic a plcation Varia les that shal beconside ed w hen using in v itro t es methods

inclu e haemat oc it, antico gulant ( y e an amount), t es sample pr p ration, t es sample ag e,

blo d/ lo d comp nent ag e, t es sample st orag e, ae ation an pH, t empe atur , pro e ran omization,

t es sample surfac ar a t o blo d v lume ratio an for dynamic s u ies, fluid flow con itions, espe ialy

flow rat e, wal shear rat e an pr s ur () Tes s shal be s art ed with minimal dela , usualy within

4 h of blo d draw, sinc some pro e ties of blo d chang e ra idly folowing cole tion Alt ernatives t o

the latt er ma be feasible if v ldat ed In some cases, the r sulting samples can also be froz n using

a pro riat e t echniq es for futur analy sis if the fr e e /thaw proc s does not afe t the analyt e being

as es ed

NOTE Clinical y relevant y es an amou ts of antico gulant may or may not b ap ropriate, depen ing on

the test sy st em an the a ility to dis ern positive an ne ative responses

When used t o ev luat e the haemocomp tibi ty ofdevic modifications, in v itro t es ing for haemolysis,

thrombus formation, plat elet an co gulation r sponses ma be as es ed an comp r d betwe n the

modified devic an the clnical y ac ept ed devic ( e A.1.4)

6.3.2 Ex vivo tests

Ex vi vo t es s shal be pe formed w hen the int en ed use of the devic is ex v i vo, e.g an ext ernal

commu icating devic Ex v ivo t es ing can also be useful w hen the int en ed use is in v ivo, e.g t o as es

the acut e r sp nse t o an implant such as a v scular graf Such use should not howeve subs itut e for an

implant t es

Ex v ivo t es syst ems ar a aia le for monit oring plat elet ad esion, embol g ene ation, f ibrinog en

dep sition, thrombus mas , w hit e-c l ad esion, plat elet consumption an plat elet activ tion

[4 ][4 ]

[4 7][5 ][54][70][7 ][80]

Blo d flow rat es can be measur d with eithe Do ple or ele troma netic flow

pro es Alt erations in flow rat es ma in icat e the ext ent an course of thrombus deposition an

emb lzation Simple thrombus buid-up can be as es ed b gros an or mic osco ic visual zation

Othe mor adv nc d an t ech ical y deman ing t ools ha e also be n used

[5 ][69][73][74][79]

6.3.3 In vivo tests

In v i vo t es ing inv lves implanting the mat erial or devic in animals Vascular pat ches, v scular

cathet ers, v scular graf s, v scular st ent , an ulo las y rings, heart v lves an cir ulat ory as is

devic s ar ex mples of devic s t est ed in v i vo Given the dive sity of blo d-contacting medical devic

a pl cations, in v ivo t es models ar ex e t ed t o be eq aly dive se, in orde t o a pro riat ely mimic

each clnical a plcation

“ Pat ency of a con uit or devic ( i.e the unimpeded flow of blo d through the devic ) is a common

measur of suc es or faiur for some in v i vo ex e iment The pe c nt oc lusion an thrombus mas

ar det ermined aft er the devic is r mo ed The t en ency of thrombi formed on a devic t o emb l z

t o dis al org ns should be as es ed b car ful gros as wel as mic osco ic ex mination of org ns

downs r am from the devic In ad ition, hist op tholo ical ev luation of the sur oun ing tis ue an

org ns is useful The kidney s ar espe ialy prone t o tra thrombi w hich ha e emb lz d from devic s

implant ed ups r am from the r nal art eries (e.g ventricular-as is devic s, artif icial heart , a rtic

pros hetic graf s)

[18 ][18 ][2 6][2 7]

Methods t o ev luat e in v i vo int eractions without t erminating theex e iment ar a aia le Art erio rams

or ima ing from intra ascular ultrasoun (IVUS) cathet ers ar used t o det ermine p t ency or thrombus

deposition on devic s Radioima ing can be used t o monit or plat elet dep sition at v rious time pe iods

in vi vo; plat elet surviv l an consumption can be used as in icat ors of blo d/devic int eractions an

pas iv tion d e t o neointima formation or prot ein adsorption

[4 ][72][79]

In some in v ivo t es sy st ems, the mat erial’ s pro e ties ma not be major det erminant of the

blo d/devic int eractions Rathe , flow paramet ers, complanc , p rosity an implant design ma be

mor imp rtant than blo d compatibi ity with the mat erial it elf As an ex mple, low flow rat e sy st ems

ma give subs antialy dife ent r sult w hen compar d with the same mat erial ev luat ed in a high

flow rat e sy st em In such cases, t es sy st em pe formanc in v i vo should car y mor importanc than in

vitro t es r sult

In v i vo t es prot ocols should contain pr cise an s an -alone se tions s ating how each t es cat eg ory

identified for t es ing, i.e haemoly sis, thromb sis, co gulation, plat elet , haemat olo y an complement

sy st em, wi beev luat ed

This annex pro ides backgroun for sele ting t es s t o ev luat e the int eractions of cardio ascular

devic s with blo d Clause 6 contains guides t o det ermine w hen t es ing is ne es ary, w hich blo d

int eraction cat eg ories might be a pro riat e for spe if ic devic s, an a ls of t es s for ev luating

blo d/devic int eractions of non-contact-, ext ernal commu icating - an implant devic s The

clas if ication of blo d/devic int eractions in A.1.2 ispro ided as b ckgrou d

A 1.2 Clas ification

A 1.2.1 Interactions which mainly afe t the devic an which ma y or ma y not ha ve an u desirable

efe t on the animal or h man ar e as folows:

a) adsorption of plasma prot eins, lpids, calcium or othe subs anc s from the blo d ont o the surfac

of the devic ;or a sorption of such subs anc s int o the devic ;

b) adhesion of plat elet , leu ocyt es or e ythrocyt es ont o the surfac of the devic , or a sorption of

their component int o the devic ;

c) formation of pseu ointima or neointima on the blo d contacting surfac an tis ue ca sule on the

surfac of the devic ;

d) alt erations in me hanical an othe pro e ties of the devic

A 1.2.2 Interactions which ha ve a p tentialy u desirable efe t on the animal or h man ar e as folows:

a) activ tion of plat elet , leukocyt es or othe c l s, or activ tion of the co gulation, fibrinolytic, or

complement pathway s;

b) formation of thrombus on the devic surfac ;

c) emb lzation of thromb tic or othe mat erial from the devic ’ s surfac t o anothe sit e within the

cir ulation;

d) injury t o cir ulating blo d c l s r sulting in anaemia, haemoly sis, leuco oenia, thromb cyt openia

or alt er d fu ction of blo d c l s;

e) injury t o c lsan tis ues adjac nt t o the devic ;

f) intimal hy e plasia or ac umulation of othe tis ue on or adjac nt t o the devic , r sulting in

r d c d flow or afe ting othe functions of the devic ;

g) adhesion an growth of b ct eria or othe infe tious ag ent on or near the devic

NOTE For items b), c)an d) a ove, some devices such as emb lization coils req ire thrombus formation to

b fu ctional

A 1.3 Ad vantag es and l mitations of animal models

Animal models pe mit the closes en use simulation of cl nical devic s prior t o actual t es ing in

h mans They pe mit contin ous devic monit oring and a sy st ematic controled inves ig tion of

imp rtant v ria les Howeve , the choic of an animal model ma be r s rict ed b siz r q ir ment ,

the a ai a i ty of c rtain spe ies an cos F or ex mple, a devic ma not be o e at ed u de the ful

rang e of clnical use con itions in an animal model d e t o anat omical lmitations It is c itical that the

inves ig t ors be min ful of the phy siolo ical dife enc s an simi arities of the spe ies chosen with

those of the h man, p rticularly those r lating t o co gulation, plat elet fu ctions an f ibrinoly sis,

an the r sponse t o pharmacolo ical ag ent such as anaes hetics, antico gulant , thromb lytic an

antiplat elet ag ent and antibiotics Be ause of spe ies dife enc s in r activity, subje t dife enc s in

r activity an v ria le r sponses t o dife ent devic s, data o tained from a single spe ies should be

int erpr t ed with caution Non-h man primat es such as ba o ns exhibit a close simiarity t o the h man

in haemat olo ical v lues, blo d co gulation me hanism an cardio ascular sy st em

[5 ]

An ad itional

adv ntag e of a non-h man primat e is that many of the immunolo ical pro es for thromb sis as ay s

develo ed for h mans ar suita le for use in primat es These pro es inclu e PF-4, β-T , FPA, T AT

an F 2 The do is a commonly used spe ies an has pro ided useful information; howeve , devic

-r lat ed thromb sis in the do t en s t o oc ur mor r adi y than in the h man, a dife enc w hich can

be viewed as an adv ntag e (as a chalenging or ac ele at ed model) w hen ev luating this complcation

Pigs an she p ar g ene aly r g rded as suita le animal models be ause of their haemat olo ical an

cardio ascular simi arities t o the h man

[71][14 ][149][1 0]

The efe t of the surgical implant proc d r

on r sult should bekept in min an a pro riat e controls inclu ed The final de ision on the use of an

animal or in v itro model ultimat ely inv lves conside ation ofthe a ai a i ty an ethical use of animals

( e ISO 1 9 3- 2), the a aia i ty and l mit of in v itro blo d models an the a plca i ty of pro e

s atis ics for sou d conclusions

[2 3][2 4][2 5][2 6]

A 1.4 Ad vantag es and l mitations of in vitr models

In v itro blo d-ex osur models ar at ractive a pro ches t o t es ing the haemocomp tibi ity of medical

mat erials an cardio ascular devic s be ause they alow

a) a oidanc of cos ly animal models,

b) high r plcation t es ing of t es o je t alongside controls and r fe enc mat erials using the same

bat ch of blo d an at he same time,

c) use of h man or animal blo d w he e flow, t empe atur an antico gulation is s an ardiz d,

d) wors case sc nario t es ing, w he e activ tion prod ct ac umulat e without clearanc b kid ey s

or lve or othe org ns an activ tion-inhibiting functions of en othel al c l s ar a sent, and

e) isolation from confoun ing fact ors as ociat ed with devic implantation/tis ue injury as ociat ed

with in v ivo usag e

Such t es ing of medical mat erials and devic s should simulat e as bes as p s ible the rang e of clnical

con itions of blo d ex osur t o the devic , sinc t es ing on blo d u de clnical y ina plca le con itions,

e.g non-clnical antico gulation ( y es or levels) and flow con itions, can make int erpr tation of r sult

diff icult Wheneve p s ible, consult prod ct IFU broch r s or common medical practic lt eratur

for a plca le antico gulant ty e() an amount (s) When a pro riat e, t es ing o e the ful rang e of

la eled use con itions for the devic should be conside ed F or ex mple, t o ev luat e me hanical

y-in uc d haemoly sis an plat elet activ tion, t es ing is oft en pe formed at the highes blo d flow rat e

F or thromb sis t es ing, the minimum la eled blo d flow rat e ma be imp rtant t o charact eriz the

safety of the devic Sinc it has be n shown that r sp nses in blo d ma dife conside a ly betwe n

v rious spe ies

[4 7][14 ][149][1 0]

the use of h man blo d is mor r lev nt t o the int erpr tation of

r sult Anothe adv ntag e in using h man blo d is that it ofe s a mor detaied ar a of t es methods,

sinc mos cont emp rary bio nalytical methods ar b sed on h man blo d component /epit opes

Conve sely, the e ar c rtain l mitations in the v lume of blo d that can be o tained from a single

h man donor Th s, the use ofblo d from a single larg e animal ma be mor practical in cases w he e

the model designed t o simulat e clnical-r lev nt con itions pr sent a larg e volume ca acity

To t es for g ene al mat erial/devic haemocompatibi ity, the clas ical Chan le lo p in v itro t es model

or modif ications the eof

[1 3][1 4][1 5][1 9][2 0][2 3]

t o impart phy siolo ical an / r q asi-phy siolo ical

flow ha e be n used Alt ernatively, blo d-mat erial (devic ) ex osur using g entle a itation ma also be

useful in somecases for ev luating the int eractions of blo d with mat erials To g ug e the impact of the

model on blo d, haemoly sis an g ene al c l blo d cou t can be monit or d t o che k for blo d normalcy

These models a pear efe tive for sc e ning s u ies, in particular for those a plcations inv lving

short-t erm blo d ex osur

A 1.5 Test protoc ols for animal testing

Thromb sis, thromb emb l sm, ble ding an infe tion ar the major det er ent t o the use an furthe

develo ment of adv nc d cardio ascular pros heses F or devic s with l mit ed blo d ex osur (< 4 h),

imp rtant measur ment ar r lat ed t o the ext ent of acut e v riation of haemat olo ical, haemodynamic

an pe formanc v ria les, gros thrombus formation an p s ible emb lsm W ith prolong ed or

r peat ed ex osur or pe manent contact (>24 h an > 0 d, r spe tively), emphasis is plac d on se ial

measur ment t echniq es that ma yield information r g rding the time course of thromb sis an

thromb embol sm, the consumption of cir ulating blo d comp nent an the develo ment of intimal

hy e plasia an infe tion In both of these ex osur and contact cat eg ories, as es ment of haemoly sis

an plat elet function is imp rtant Thrombus formation ma be gr atly influenc d b surgical

t ech iq e, v ria le time-depen ent thromb lytic an emb lc phenomena, supe imp sed devic

infe tions an p s ible alt erations in ex osed surfac s, e.g intimal hy e plasia, fibrotic enca sulation

an en othelal zation Imp rtantly, antico gulation ty e() an amount (s) can ha e a profou d

imp ct on r sult F or ex mple, at cl nicaly r lev nt levels, antico gulation an antiplat elet drugs ma

subs antialy r d c or a ol sh plat elet, co gulation an thromb tic r sp nses

The conseq enc s of the int eraction of artif icial surfac s with the blo d can rang e from gros

thromb sis an emb l zation t o subtle efe t such as ac ele at ed consumption of element inv lved

in normal haemos asis The latt er ma be cl nicaly insignificant, e.g plat elet consumption b the

devic could be so smal that it does not afe t the t otal plat elet cou t Alt ernatively, a devic with a

larg e surfac ar a could lead t o depletion of plat elet or plasma co gulation fact ors such that the t otal

plat elet cou t ma be signif icantly afe t ed an normal haemos asisma be ome alt er d

R eg rdles of the animal model used an the p rticular t es cat eg ory un e ev luation, i.e haemoly sis,

thromb sis, co gulation, plat elet , haemat olo y an complement sy st em, the in v i vo s u y prot ocol

should pro ide sufficient detai in the methods and c it eria t o be used for ev luation for each t es

cat eg ory un e inves ig tion A r trospe tive r p rt on r sult for a p rticular t es cat eg ory, without

sup orting original plans within the prot ocol, is oft en conside ed u ac epta le as r gulat ory

submis ion documentation

A.2 Cannulae used for direc t vasc ular ac c es and c annulae used for indirec t ac c es

The t erm “can ulae” has be n g ene aly used in two rathe dife ent clnical a plcations In one

a plcation, cann lae ar inse t ed dir ctly through the skin an int o one or mor major blo d ves els

This is done t o pro ide contin ous an dir ct high-volume ac es t o blo d F or ex mple, this ty e of

larg e-diamet er cann lae is used d ring cardio ulmonary b p s surg ery as a l mit ed-ex osur ac es

devic that sh nt blo d t o an from the b dy for blo d o y g enation Cann la t es ing, in this ex mple,

should take plac using ex osur con itions that closely r plcat e cl nical use, as such devic s can

p t ential y in uc some alt eration in the levels of cir ulating blo d c ls as wel as inc ease fact ors in

the co gulation or complement sy st em The particular r sp nseis oft en multifact orial as it depends on

a v riety of fact ors such as implantation sit e, inse tion t ech iq e, subje t fact ors an antico gulation

r gimen The t erm cann lae has also be n used t o desc ibe much smal e diamet er tubes that ar

inse t ed only subcutaneously, an ma be used for l mit ed (< 4 h) or prolong ed (< 0 d) in ir ct

ex osur t o blo d These can ulae, for ex mple, ar used for infusion of insuln from drug pumps an

in subcutaneous sensing for blo d glucose levels These lat er ty e of cann lae, lke othe in ir ct blo d

p th devic s ( e 5.2.2), g ene al y r q ir les t es ing than devic s with dir ct contact with cir ulating

blo d ( e 5.2.3 an 5.3)

A 3 Catheter s and g uide w ir es

Mos of the t es s conside ed u de blo d-contacting cann lae ar r lev nt t o the s u y of blo

d-contacting cathet ers an guide wir s The location or plac ment of cathet ers in the art erial or venous

sy st em can ha e a major efe t on blo d/devic int eractions It is advised that simultaneous control

s u ies, using a clnical y a pro ed devic of simiar dimensions an mat erial(), be pe formed using a

contralat eral art ery or vein Car should be taken not t o s rip of thrombus up n cathet er with rawal

Ev luation of the devic in s itu ma pe mit as es ment of the ext ent t o w hich intimal or entranc

sit e injuries contribut e t o the thromb tic proc s In g ene al, Do ple blo d flow measur ment

ar mor informative than angio ra hy A venous or art erial implant model with antico gulation

pe tinent t o the clnical a plcation ma be a useful t ool for ev luating devic blo d-contact r sponses,

p rticularly w hen as es ing a new devic mat erial or a co ting develo ed t o pr sent anti-thrombog enic

pro e ties

[143][161][162][163]

S e C.3 Alt ernatively, an a pro riat e in v itro model ma be mor sensitive

t o det ecting such mat erial surfac dife enc s

In cases w he e antico gulation is caled for, the rationale for the ty e an level of antico gulation

used in t es ing should be b sed up n the clnical a plcation, yet be a le t o pro ide suff icient evidenc

that the t es is a le t o dis inguish betwe n positive an neg tive r sp nses F r ex mple, folowing

simple dose-r sp nse kinetics, the thromb r sis anc of a medical devic hep rin co ting can be

complet ely masked b normal (cl nical) levels of solution heparin antico gulant Howeve , un e

a r d c d/chalenging level of solution hep rin, the efe tivenes of the hep rin co ting t o r d c

thrombus formation be omes mor a p r nt In cases w he e the a plcation ma not inv lve use of

antico gulation, t es ing should be cond ct ed without antico gulation

Valdation information for t es ing with a spe if ic ty e an level of antico gulation should demons rat e

the a i ity t o disc rn betwe n p sitive and neg tive r sp nses

A 4 Ex trac orpor eal blood ox yg enators, haemodial ysis/haemofi tration devices,

donor and therapeutic apher esis equipment , devic es for adsorption of specific

substances from blood

The blo d r sp nses t o cardio ulmonary b p s can be signif icant an acut e Many v ria les such

as use of blo d suction, comp sition of blo d-pump priming fluid, hy othe mia, blo d contact

with air an time of ex osur influenc t es v lues Emb l in out low l nes ma be det ect ed b the

pe iodic plac ment of blo d filt ers ex v ivo or the use of ultrasou d or othe non-inv sive t ech iq es

Thrombus ac umulation can be dir ctly as es ed d ring b p s b monit oring pe formanc fact ors

such as pr s ur dro ac os the o yg enat or an o y g en trans e rat e An acq ir d transient plat elet

dy sfu ction as ociat ed with sele tive alpha gran le r lease has be n o se ved in p tient on

cardio ulmonary b p s

[1 8]

othe t es s of plat elet fu ction an r lease ar p rticularly useful

Complement activ tion is caused b b th haemodialy se s an cardio ulmonary b p s eq ipment

Clnical y significant pulmonary leucos asis and lu g injury with dy sfu ction can r sult

[5][1 ][16][1 9]–

[14 7]

F or these r asons, it is useful t o q antify complement activ tion or leuko enia with these devic s

S e also An ex E

The a eutic a he esis eq ipment an devic s for adsorption of spe ific subs anc s from the blo d,

be a use of their high surfac -t o-v lume ratio, can pot entialy activ t e complement, co gulation, plat elet

an leukocyt e p thway s Ex mination of blo d/devic int eractions in these an any othe high surfac

ar a devic s should folow the same principles as for extracorp r al o yg enat ors an haemodialy se s

A 5 Ventricular-as ist devic es and total artificial hearts

These devic s can in uc conside a le alt eration in v rious blo d comp nent F act ors contributing t o

such efe t inclu e the larg e for ign surfac ar a t o w hich blo d is ex osed, the high flow r gimes an

the r gions of dis urbed flow such as turbulenc or separat ed flow Tes s of such devic s ma inclu e

measur ment of haemoly sis, thrombus formation, f ibrin formation, thromb emb l zation, thrombin

g ene ation, plat elet surviv l an activ tion, complement activ tion and close monit oring of lve ,

r nal, pulmonary an c ntral ne vous sy st em efe t A detaied p tholo ical ex mination at surgical

r triev l is an imp rtant component of the ev luation

[2 6][2 7]

A 6 Heart val ve prostheses

Inv sive, non-inv sive and in v itro hydrodynamic s u ies ar imp rtant in the as es ment of

pros hetic v lves

One of the mos efe tive meansof sc e ning for pros hetic v lve dy sfunction is auscultation

[18 ]

Two-dimensional an M mode e hocardio ra hy makes use of ultrasonic radiation t o form imag es of the

heart R efle tions from mat erials with dife ent acous ic impedanc s ar r c ived an proc s ed t o

form an imag e The s ructur of pros hetic v lves can be ex mined Me hanical pros hesesemit s rong

e ho signals an the mo ement of the oc lu e can usualy be clearly imag ed Howeve , the q alty

of the imag e ma depen up n the particular v lve being ex mined Echocardio ra hy can also be

useful in the as es ment of fu ction of tis ue-de ived v lve pros heses Veg etations, thrombus an

evidenc of thickening of the v lve leaflet ar elucidat ed Using conventional an colour flow Do ple

e hocardio ra hy, r gurgitation can be identified an semi-q antif ied

[2][185][18 ][18 ]

Measur ment of plat elet surviv l an a gr g tion, blo d t es s of thromb sisan haemoly sis, pr s ur

an flow measur ment , an aut opsy of the v lve an adjac nt tis ues ar r commen ed

[2 5][2 6]

A 7 Vascular g rafts

Both p rous an non-p rous mat erials can be implant ed at v rious locations in the art erial or venous

sy st em The choic of implantation sit e is det ermined larg ely b the anat omical conside ations of

the model an the cl nical sit e of use Pat ency of a given graf is enhanc d b larg er diamet er an

short er length Pat ency can be document ed b palpation of dis al pulses in some locations an b

pe iodic angio ra hy Ultrasoun , MRI an P T ma also be useful S rial measur ment of plat elet

count, plat elet r lease cons ituent , f ibrinog en/fibrin degradation prod ct an activ t ed co gulation

prot eins also ar r commen ed A ut opsy of the graf an adjac nt v scular segment for v scular

tis ue r sp nses can pro ide v lua le information A sy st ematic ev luation of longitu inal an c os

-se tional se tions of pro imal an dis al anast omoses an r pr sentative midgraf r gions is ne es ary

for a thorough ev luation of the devic

[4][2 5]

As with many v scular devic s, a pro riat e clnical

antico gulation r gimens ar c itical t o devic function an pe formanc

A 8 IVC fi ters, stents and stented g rafts

These devic s can be s u ied b angio ra hy an ultrasonic radiation Othe t ech iq es useful for

v scular graf ev luation ( e A.7)ar a pro riat e he e as wel

2 5]

The g ene al principles an scientific b ses of the mor c ommo ly used t es s t o ev luat e the cat eg ories

of haemoly sis, thromb sis, co gulation, plat elet , haemat olo y an complement sy st em ( e 6.2)

ar desc ibed in B.1 t o B.3 S e Annex es C, D an E for furthe information on the t es cat eg ories of

thromb sis, haemoly sisan complement

A dditional, albeit les common methods, that ma be of furthe v lue in the ev luation of p rticular

blo d/devic int eractions ar desc ibed in Annex F Be ause of biolo ical v ria i ity and t ech ical

l mitations, the ac uracy an pr dictivity of many of these t es s r q ir car ful att ention t o

methodolo y an caution in int erpr tation of r sult An ex G ls s t es s w hich ar not r commen ed

B.4 pr sent methodolo y conside ations for t es ing of plasma fact ors spe if ic t o co gulation, plat elet

an leucocyt e activ tion an complement activ tion using ELISA (or othe simi ar) t ech iq es

Al r fe enc s in the biblo ra hy desc ibein mor detai an give ex mples of v rious s an ards, t es s

an models for conside ation

B.1.2 In vitr ver sus e x viv ver us in viv testing

B.1.2.1 A hos of in vitro, e x viv an in viv models ha ve be n used ex tensively to estimate blo

d-mate ial inter actions

[1]–[3 ][42]–[14 ][1 7]–[2 7]

It isa pr opriate to sele t he model mos suita le for the

devic a plcation an tes o je tive and to consult ISO 1 9 3-12 r egarding pr ope sample pr ep r ation

and control g r oup considerations

No single in v itro, ex v ivo or in vi vo model wi be a pro riat e for al a plcations Th s, the

a pro riat enes of the model t o the a plcation un e conside ation should be jus if ied

In v ivo t es s pr sent a mor r als ic en -use simulation, yet ar complcat ed b fact ors such as:

— choic of a pro riat e animal model;

— int erspe ies and int ersubje t v ria i ty in r sp nses

[4 7][71][14 ][149][1 0]

— scar ity of spe ies-spe ific comme cial t es kit for common in icat ors of thrombosis an

co gulation

[58][5 ][60]

— height ened cos s an ethical an s atis ical conc rns inv lved in using animal models

B.1.2.2 Consult with ve tical s an ar ds for pr efe red models

[1]–[41]

S e also Refer enc [18 ] for

the juvenie she p as an ac elerated model to s u y bio-pr os hetic v lve calcification, Refer enc [18 ]

for the ad lt pig or she p to inves igate tr ansv scular-plac d v lves and sur g icaly implanted v lves,

Refer enc s [2 7] to [2 1] for the ad lt canine an she p femoral r eplac ment models used in tes ing on

smal an lar g e diamete v scular g r af model, an Refer enc s [2 2] to [2 5] for the por cine cor onary

model used extensively to inves igate s ent desig ns

B.1.2.3 A s desc ibed in othe p rt of ISO 1 9 3, car efuly con ucted invitro tes s ofe v ld scr eening

to ls to as es the biolog ical safety of medical devic s and mate ials

Imp rtant fact ors in an in v itro model that r q ir spe ification ar

— v lume of w hole blo d in the b sic t es sy st em, e.g tube, lo p, or othe model,

— blo d ex osur time(),

— blo d t empe atur ,

— blo d flow condition,

— antico gulant ty e an level,

— ex osur ratio, i.e ratio of mat erial/devic surfac ar a (cm

NOTE 1 The “blo d e posure p ase” of an in v itr investig tion req ires a precise def inition of the e posure

con itions of the test material/device to blo d T e closer the test con itions mimic the clinical ap lication,the

gre ter the predictivity b comes of the model an the response(s) b ing evaluated

NOTE 2 A “testing p ase” folows the e posure p ase where specif ic tests are con uct ed on the e posed

blo d, blo d plasma or the material/device itself A test in the t esting p ase is usualy targ ted at one or more of

the g neral cat eg ries, i.e ha moly sis, thromb sis, co gulation, platelets, ha mat olo y an complement system

B.2 an B.3 ex mine the common methods used t o as es the main cat eg ories of blo d-mat erials /devic

int eraction ( e Ta le 2)

B.2.1 Gros anal ysis — Retr ieval and examination of devic e and autopsy of distal or gans

Gros analy sis should alway s be inclu ed as p rt of a b sic devic ev luation, as this segment of a devic

ev luation is of c ntral imp rtanc in ev luating the in v i vo biolo ical r sp nses t o implant ed devic s

The dis ribution, visible siz an natur of c l ular an prot einac ous deposit , an any emb l, can

bes be det ermined b a car ful an detai ed gros ex mination Pro osed proc d r s ha e be n

publ shed

[7][2 5][2 6][2 7]

The rationale behin ne ro sy of dis al org ns is t o ex mine for dis al efe t ( uch as emb l) of

implant ed devic s The imp rtanc of this analy sis v ries with devic a plcation an is r s rict ed

t o a plcations w he e risk for thromb emb l sm or mat erial/devic emb lzation, for ex mple with

me hanical heart v lves an intra-a rtic b lo n pumps, is int ermediat e t o high

[2 6]

In this ty e of inves ig tion, low- an / r high-ma nification high-r solution colour f ilm or digital

imag es at key p int of int er s (of the devic , an the sur ou ding tis ues, et c ) ar taken an la eled

a pro riat ely

B.2.2 P erc entag e oc clusion, surface ar ea c overed by thr ombus and thr ombus-fr ee

surfac e area

Pe c ntag e oc lusion ma be q antitatively as es ed d ring the in-lfe p rtion of the s u y using

contras radio ra hy an ultrasono ra hy ima ing t echniq es Pe c ntag e oc lusion can also be

visualy as es ed aft er an implant ed devic has be n r mo ed The pe c ntag e of oc lusion ma be a

measur of the seve ity of the thromb tic proc s in a con uit Howeve , lack of oc lusion does not

ne es ari y elminat e the exist enc of a thromb tic proc s , sinc thrombi ma ha e emb lz d or be n

dislodg ed befor pe c ntag e oc lusion is measur d Oc lusion ma be caused not only b thromb sis, but

also b intimal hy e plasia, espe ial y at pe i-anast omotic sit es in v scular grafs Th s, a sup orting

surfac ar a co e ed b thrombus an thrombus-fr e surfac ar a ar semi-q antitative or q antitative

t es s that can be used on a comp rative basis with t es an / r control devic s

B.2.3 Lig ht microsc opy

By this t ech iq e, information can be o tained r g rding the density of c l s, pr senc of c lular

a gr g t es, comp sition of enca sulating tis ue, int ensity of for ign b dy r sponse an thrombus

or f ibrin ad e ent t o mat erials The ev luation of g eo ra hic dis ribution of these deposit on the

mat erials or devic is also p s ible The method is semi-q antitative

F or p lyme ic or biolo icaly-de ived mat erials an devic s, p raff in wa embed ing methods an

spe ial s ains ma be used t o as es the devic -biolo ical int erfac

F or metal an c ramic mat erials an devic s, mor so his icat ed hard plas ic embed ing an se tioning

t ech iq es ar useful t o ca tur the intact mat erial/devic -biolo ical int erfac

[2 7]–[2 1]

B.2.4 Scanning electron micr osc opy (SEM)

F or SEM, rationale an int erpr tation ar the same as for lght mic osco y ( e B.2.3) This method has

the adv ntag e o e lght mic osco y of pro iding gr at er detai a out fine s ructur of component

being ex mined Q uantitative conclusions r q ir sufficient r plcat e det erminations t o es a lsh

degr e of r prod cibi ty This ty e of mic osco y bes r fle t w hat can be se n at surfac s Cros

-se tional analyses can also be used t o sup ort the surfac o se v tions if ad itional detai s r g rding

the c l and thrombus surfac int eractions ar informative

[70][71][143][2 5][2 6]

The morpholo ical

ev luation of plat elet an leukocyt e activ tion, fibrin an thrombus formation aft er blo d or blo d

comp nent (e.g plat elet-rich plasma) dynamic ex osur with comparison t o r fe enc controls is

v lua le

[143][173]

B.3.1 Ha mol ysis — Methods for testing

Haemoly sis is r g rded as a significant sc e ning t es be ause an elev t ed in vi vo plasma haemo lo in

level is a normal an ma be in icative of an u de lying haemo atholo y or v scular pro lem Pro e ly

pe formed, an elev t ed plasma haemo lo in level in icat es haemoly sis, i.e the r lease of cont ent

of r d blo d c l s (RBCs), an ma r fle t e ythrocyt e membrane fra i ty or damag e t o RBCs In the

as es ment of blo d-mat erial/devic int eractions, haemoly sis ma r sult d e t o:

a) dir ct blo d contact with the mat erial()/devic surfac ( ) (mat erial-in uc d);

b) in ir ct contact from ex osur t o extracta le chemicals from the devic mat erials (mat erial

in uc d);

c) ex osur t o turbulenc and elev t ed ( i.e non-phy siolo ical) shear s r s es from the devic

o e ation (me hanicaly-in uc d)

S e Annex D for mor ext ensive detais on t es ing for haemolysis

B.3.2 Coag ulation — Methods for testing

B.3.2.1 General

The co gulation cascade has two paralel p thway s, the c ontac t ac ti vation p thwa (he intrinsic

p thwa )an the ti s s ue f c tor p thwa ( he extrinsic p thwa ) that me g et o form a common pathwa

The latt er inclu es the prot ein thrombin, w hich cataly ses the formation of fibrin, a main comp nent

of a thrombus Whie it is k own that the primary p thwa for the initiation of blo d co gulation is

the ti s s ue f c tor p thwa , co gulation as ociat ed with blo d contacting devic s an mat erials oc urs

through the contact activ tion p thwa The p thway s themselves ar a se ies of r actions in w hich

co-fact ors t o be ome active comp nent in a cascade of activ tion event The r actions culminat e in

the formation of active thrombin that then catalyses the formation of f ibrin C a ulation fact ors ar

g ene aly in icat ed b R oman n me als, with a lower ase “a” a pen ed t o in icat e the active form S e

Figur B.1

As es ment of co gulation activity, i.e the degr e of chang e in blo d levels ofprot eins leading t o

thrombin an f ibrin formation ( e Figur B.1), has long r led on clnical as ay s that measur plasma

levels of key prot eins in the co gulation cascade Normal r s ing (homeos asis) levels of co gulation

activity ar wel es a lshed, as ar some elev t ed levels o se ved in v rious clnical co gulo athies

The pr sumption for such t es ing with medical devic s is that a pro riat e mat erials and devic designs

should not be as ociat ed with ex ces ive co gulation activity that could bring risk t o the p tient High

levels of co gulation activity ma be an in icat or of a highe t en ency for the mat erial or devic t o

in uc or be as ociat ed with acut e thrombosis or thromb emb l sm To measur co gulation activity,

clnical la orat ories oft en r ly up n t es kit that use common enzyme-lnked immu osorbent as a

t ech iq e In a b sic s u y, w hich ma be eithe in v ivo or in v itro an wi depend up n a ai a i ty

of a pro riat e antib dies t o spe ies-spe if ic targ et co gulation prot ein epit opes, blo d samples ar

r trieved un e defined con itions an pr p r d an analy sed pe as a ins ructions Ty ical def ined

con itions or fact ors important in an in v itro model ar desc ibed in B.1.2.3 C mparison of r sult t o

a pro riat e controls such asneg tive controls (e.g baselnelevels or no mat erial/devic ex osur ) an

r sult on a pr dicat e devic ()/mat erials s) isc itical C a ulation activity in blo d that is s atis ical

y-significantly an biolo ical y-signif icantly highe than controls ma be an in icat or of a mat erial/devic

design that pr sent a highe risk of co gulation-r lat ed complcations Ex mple co gulation activ tion

prot eins for w hich comme cialy-a aia le ELISA kit ar a aia le inclu eT AT ( hrombin-antithrombin

complex es), F.1.2 (prot ein fra ment r leased from prothrombin up n formation of thrombin) an FPA

(prot ein fra ment r leased from f ibrinog en up n formation of fibrin)

Prot eins in icating co gulation activ tion g ene aly exhibit an initiation, pro a ation an t ermination

phase

[56][57]

This r fle t the initial formation r action(), a cascade / fe db ck ampl f ication pe iod

an a slowdown/deactiv tion pe iod w he e c itical pr cursors ma be consumed or the measur d

prot ein deactiv t ed b neg tive control fe db ck prot eins Th s, order-of-ma nitu e dife enc s in

levels of co gulation activ tion prot eins ar t o be ex e t ed o e time Conseq ently, an imp rtant fact or

t o conside is w hen the activ tion phase actualy oc urs d ring the time of the mat erial/devic -blo d

contact F r ex mple, the imp ct of t es mat erials w hen mix ed with blo d ma be q it e dife ent at

each phase In ad ition, as co gulation prot ein activ tion isg ene aly pro ortional t o blo d-contacting

surfac ar a, surfac ar a ( SA) ofa devic or devic mat erial can be ve y influential on r sult F or

this r ason it is imp rtant t o spe ify the t es SA-t o-blo d-v lume ratio (ex osur ratio) in each s u y

If p s ible, the ex osur ratio ma be tr at ed as a v ria le t o aid in u de s an ing the spe ificity of

the mat erial efe t Ex osur ratios of 3,0 cm

2

t o 6,0 cm

2

/ ml blo d (b sed on devic thicknes ) ar

consist ent with ISO 1 9 3-1 Othe ex osur ratios such as 1,5 an 2,0 times this ratio ma be worth

conside ing as highe surfac ar as wi theor ticaly inc ease the sensitivity of the co gulation

r sp nses t o the t es mat erial

The e wi l be a phy sical lmitation on the amou t of t es mat erial that can be t est ed d e t o the v lume

of the t es sy st em, e.g a t es tube, and the targ et ex osur ratio In this case, it ma be a pro riat e t o

use cut se tions of devic mat erials If the devic contains mor than one mat erial, the pro ortion of

each in the complet e devic should be maintained Car should also be made t o a oid introd cing cut

se tions that r sult in ex osur of significant amou t of non-blo d contact surfac s

Natural y, the e ar a n mbe of me hanisms that ha e ev lved t o keep the co gulation cascade in

che k One of those me hanisms involves the prot ein antithrombin Antithrombin is a se ine prot ease

inhibit or that can bin t o an deactiv t e the se ine prot eases thrombin, FIXa, FXa, FXIa an FXI a

Whie antithrombin is cons antly active, it int eraction with hep rin alt ers it conformation, w hich

gr atly ac ele at es it rat e of in ibition of the prot eases

NOTE E ISA testing for blo d co gulation factor represents the “testing p ase” dis us ed in B.1.2, i.e

for testing on the blo d samples procured flow in in v itr or in v i vo blo d e posure t o the medical device or

material

Many s an ard co gulation as ay s ar designed t o det ect clnical co gulation disorde s w hich r sult

Prot ocols for ev luating blo d/devic int eractions shal be modified a pro riat ely t o ev luat e

ac ele at ed co gulation in uc d b biomat erials

Int eractions betwe n the co gulation and complement sy st ems ar r co niz d

[143]–[14 7]

Figure B.1 — Coa ulatio cas ade

B.3.2.2 Thrombin-antithrombin (TAT), F 2 and fibr in (FPA ) E ISA as ays

These ELISA as ay s that dir ctly r fle t thrombin (T AT, F 2) and f ibrin (FPA) formation ar

comme cialy a ai a le The output is a q antitative es imat e of the amou t of thrombin pr sent an

the amount of fibrin being formed, b th of w hich ar r fle tive of the level of co gulation activity

taking plac an ma be r fle tive of thromb sis taking plac S e B.4 for detais on g ene al ELISA

methodolo y

B.3.2.3 P ar tial thr omb plastin time (PTT)

Thepartial thromb plas in time is theclot ing time of r calcified citrat ed plasma up n the ad ition of

partial thromb plas in w hich does not contain an activ t or Partial thromb plas in is a phospholpid

suspension usualy extract ed from tis ue thrombo las in, the homog enat e from mammal an brain

or lu g Short ening of the PTT folowing contact with a mat erial u de s an ard conditions in icat es

activ tion of the intrinsic co gulation pathwa of blo d co gulation Hep rin an othe antico gulant

cause a prolong ed PTT S e R efe enc [2 ]

R eag ent for t es s b sed on the activ t ed p rtial thromb plas in time (APTT)inclu e an activ t or, such

as ka ln, c lt e or ela ic acid R eag ent with such activ t ors should be avoided w hen as es ing the

efe t of blo d-contacting devic s or devic mat erials be ause they mask the co gulation caused b

mat erials or devic s

In co gulation t es ing of medical mat erials an devic s, it is the devic or mat erial it elf that se ves

as the activ t or of co gulation Ap ro riat e positive an neg tive control mat erials should be used

w heneve a aia le A neg tive control, the blo d plasma it elf without the mat erial/devic , should be

inclu ed

B.3.3 Platelets — Methods for testing

B.3.3.1 General

As es ment of plat elet an their s at e of activ tion has be n desc ibed throughout the l t eratur , e.g

se R efe enc s [6 ]t o [94]as a p rtial ls Howeve , for blo d-contacting medical devic s an mat erials,

the mos commonly used methods ha e argua ly be n simple counting of plat elet an measur ment

of plat elet degran lation prot eins folowing controled ex osur of medical devic s or mat erials t o

blo d Normal r s ing (homeos asis) levels of plat elet an degran lation prot eins ar wel es a lshed

( e comme cial ELISA kit prod ct lt eratur ), as ar some a normal levels o se ved in v riousclnical

thromb cyt op thies The pr sumption for such t es ing with medical devic s is that a pro riat e

mat erials an devic designs should not be as ociat ed with ex ces ive plat elet consumption an / r

activ tion that could bring risk t o the p tient High levels of plat elet los an / r degran lation ma be

an in icat or of a t en ency for the mat erial/devic t o in uc or promot e these con itions w hich can give

rise t o complcations of ble ding or thromb sis To measur plat elet cou t an plat elet degran lation,

counting is pe formed using a routine dife ential c l cou t er an the degran lation is as es ed using

s an ard enzyme-lnked immunosorbent as ay s (ELISAs) for wel-r co niz d plat elet alpha-gran le

prot eins Clnical la orat ories oft en r ly up n t es kit that use common enzyme-lnked immu osorbent

as a t ech iq e t o measur the alpha-gran le prot eins In a b sic s u y, w hich ma be eithe in v ivo or

in v itro an wi depen up n a ai a i ity of a pro riat e antib dies t o spe ies-spe ific targ et plat elet

gran le prot ein epit opes, blo d samples ar r trieved un e defined con itions an pr p r d an

analy sed pe as a ins ructions Ty ical defined con itions or fact ors imp rtant in an in v itro model

ar desc ibed in B.1.2.3 Comp rison of r sult t o a pro riat e controls such as neg tive controls (e.g

b selne levels or t es sy st em with no mat erial/devic ex osur ) an r sult on a pr dicat e devic ( )/

mat erials s) is c itical Plat elet cou t de r ases an blo d degran lation prot ein inc eases that ar

s atis icaly-signif icantly an biolo icaly-significantly dife ent than controls ma be an in icat or of

a mat erial/devic design that pr sent a highe risk for plat elet consumption an activ tion Ex mple

alpha-gran le prot eins for w hich comme cialy-a aia leELISA kit ar a ai a le inclu e

— PF4 (plat elet fact or 4, a 7 -amino acid prot ein that bin s with high aff inity t o hep rin; PF4 major

phy siolo ic role a pears t o be neutralzation of hep rin-lke mole ules on the en othel al surfac of

blo d ves els, the eb in ibiting local antithrombin I I activity an promoting co gulation), an

— βTG (beta-thromb glo ul n, a chemokine for f ibro las s and neutro his)

NOTE Thrombin from the co gulation cas ade is a potent plat elet a onist that can re dily cause plat elet

de ran lation Th s, high levels of thrombin wil cor elate with high levels of plat elet de ran lation

As in co gulation, plat elet consumption (los ) is g ene aly se n t o be afe t ed b blo d-contacting

surfac ar a Th s, surfac ar a ( SA) of a devic or devic mat erial can influenc plat elet count data

F or this r ason, it is imp rtant t o spe ify the SA-t o-blo d-volume ratio (ex osur ratio) in each s u y

If p s ible, the ex osur ratio ma be tr at ed as a v ria le t o aid in un e s an ing the spe ificity of

the mat erial efe t Ex osur ratios of 3,0 cm

2

t o 6,0 cm

2

/ ml blo d (b sed on devic thicknes ) ar

consist ent with ISO 1 9 3-1 Othe ex osur ratios such as 1,5 an 2,0 times this ratio ma be worth

conside ing as highe surfac ar as wi l theor tical y inc ease the sensitivity of the plat elet r sp nses

t o the t es mat erial

Plat elet activ tion is a proc s that oc urs o e a time (min t es t o hours) an is r co niz d t o

be pot ential y r ve sible up t o a point, or onc initiat ed, it can pro r s non-r ve sibly t o the p int

of pr senting signif icant sha e deformation, los of cyt oplasmic cons ituent an shed ing of

mic o articles and complet e des ruction This proc s is depen ent on s imulus ty e an amou t

The ear n me ous k own p t ent timulant , caled plat elet ag onis s, ex mples of w hich ar thrombin,

ADP an colag en F or ign surfac s themselves, such as blo d-contacting medical devic s, can also

beha e lke ag onis s sinc they can cause thrombin g ene ation In ad ition, plat elet can adhe e t o

these surfac s, r main ad e ed or detach in activ t ed or non-activ t ed s at es an g o through sha e

chang es leading t o plat elet des ruction Th s, as es ing the o e al s at e of plat elet activ tion at any

point in time ma benefit from the use of ag ent that help t o “ar es ” the plat elet in their phy sical

an biochemical s at e at a particular point in time C nseq ently, if plat elet as es ment can ot be

made immediat ely aft er r mo ing the t es mat erial or devic from the t es sy st em, a n mbe of ag ent

ha e be n sugg est ed t o count eract furthe plat elet activ tion an t o s a i z plat elet

Ex mples of these ag ent ar acid citrat e dextrose (ACD ), citrat e, theo hy ll ne, adenosine, dip ridimol

(CT AD ) an othe plat elet-s a i izing r ag ent , such as Thombo ix ™

1)

To the ext ent that such s a i iz rs

can minimiz art efactual plat elet activ tion, an not o scur int erpr tation of biomat erial-spe if ic

plat elet activ tion r sponses, use of such s a i iz rs is worth conside ation, if v l dat ed t o confirm that

r lev nt en p int ar not adve sely impact ed

B.3.3.2 Platelet c ount

It is imp rtant t o det ermine the plat elet cou t

[45][1 1]

be ause of the key role plat elet ha e in pr venting

ble ding an in the g ene al proc s of thromb sis A significant dro in plat elet cou t of blo d ex osed

t o a devic can be ca used b plat elet ad esion, plat elet a gr g tion, plat elet seq es ration (for ex mple

in the sple n) or thrombus formation on mat erials or devic s A r d ction in plat elet cou t d ring use

of an implant ed devic ma also be caused b ac ele at ed des ruction or r mo al of plat elet from the

cir ulation Various antico gulant ma be suita le for en me ating plat elet

[1 1]–[1 6]

Blo d cole tion t ech iq es should be r prod cible Plat elet can be ome hy e active /activ t ed un e

a v riety of con itions, inclu ing impro e blo d cole tion Tes s such as plat elet a gr g ometry an

flow cyt ometry ma be conside ed t o ve ify normal plat elet r activity an activ tion

B.3.3.3 Platelet acti vation: Platelet g ranule-rele se pr oteins beta-thromboglob ln (ß-T G) and

platelet factor 4 (PF ), thr omboxane B2 (T x B2) and platelet mor pholog ical chang es

The use of c rtain mat erials or devic s ma ca use plat elet activ tion, w hich can r sult in the folowing:

a) r lease of plat elet gran le subs anc s, such as βTG, PF4, TxB2 an se ot onin;

b) alt er d plat elet morpholo y;

c) g ene ation of plat elet mic o articles

A ctiv t ed plat elet ar pro-thromb g enic Plat elet activ tion can be ev luat ed b v rious means, such

as mic osco ic (lght an ele tron mic osco y) ex mination of morpholo y of plat elet ad e ent t o the

mat erial or devic and measur ment of βTG, PF4 an TxB2 r leased from activ t ed plat elet

βTG and PF4 ar prot eins that ar st or d in alpha-gran les of plat elet an r leased in larg e amou t

aft er plat elet activ tion

[85][8 ][8 ][1 6]

Both of these prot eins can be as es ed b comme cialy-a aia le

ELISA as ay s Inc eases in plat elet activ tion can oc ur through multiple p ths as ociat ed with medical

devic s and mat erials The device /mat erial it elf ma be plat elet activ ting, turbulenc an ex ces ive

shear for es can cause plat elet activ tion an plat elet activ tion can be ca used b p t ent ag onis s such

as thrombin w hich ma form as a r sult of thrombosis as ociat ed with the mat erial/devic or local

injury High levels of TxB2, also measura le b ELISA, in icat e high levels of it pr cursor comp u d

thromb x ne A2, a p t ent plat elet ag onis thought t o be prod c d b activ t ed plat elet ; TxB2 is

also thought t o be r la le spe ies-in ependent marker of plat elet activ tion S e B.3.3.1 an B.4 for

detai s on g ene al ELISA methodolo y It ma also be v lua le t o as es plat elet activ tion through

the ev luation of the morpholo ical chang es plat elet un e g o w hen activ t ed on a mat erial/devic

surfac

[70][71][173]

B.3.4 Ha matolog y — Methods for testing

B.3.4.1 Complete bloo c ount (CBC)

The ele tronic complet e blo d count analy sis (oft en r fe r d t o as CBC) is a vital t es used eve y da

in hospital haemat olo y la orat ories It primary purp se is t o q ickly an ac urat ely en me at e the

conc ntration of the v rious c l p pulationsin thep tient, w he e a normal r adingscan pro ide early

an vital information on a hos of pot ential disorde s The CBC is used t o det ermine the n mbe or

1) T omb Fix

M

is an example of a suita le pr od ct a vaila le commer cialy T is information is g iven for the

pro ortion of w hit e an r d blo d c ls in the b dy The analysis inclu es plat elet counting In analy ses

on blo d-mat erial/devic int eractions, CBC data pro ides b sic information on the imp ct of the

device /mat erial int eraction with formed blo d element Count of plat elet and leukocyt es pr - an

p s -blo d ex osur t o mat erial/devic ar v lua le in ded cing the los of activ t ed plat elet an

leukocyt es taken up in clot formation b thromb g enic surfac s an the efor pro ide an es imat e of

the surfac ’s thromb g enic p t ential

24]

B.3.4.2 Leukocyte acti vation

Leukocyt e activ tion can be det ermined b mic osco ic ex mination of the devic surfac for activ t ed

leukocyt es A simple mor q antitative method involves useof a comme cial ELISA as a t o det ermine

the amount of p lymorphon clear leukocyt e (PMN) elas ase r leased t o plasma fol owing activ tion

from int eraction of a mat erial or devic with blo d Anothe a pro ch b sed on the principle that

thrombi ad e ing t o mat erial wi contain a larg e n mbe of plat elet an leu ocyt esinv lvesas es ing

the de r ase in their count in blo d

[24]

B.3.5 Complement system — Methods for testing for C3a and SC5b-9

The complement sy st em r sides in blo d plasma in the form of a biochemical cascade that fu ctions

as a defenc me hanism designed t o sup lement or “complement” the a i ity of antib dies t o clear

p thog ens from the b dy It is a p rt of the immune sy st em r fe r d t o as the “in at e immu e syst em”

He e, u lke antib dy prot ection, the r sp nse activity is neithe acq ir d nor ada ta le o e time

The complement sy st em forms a fun amental lne of defenc that works alongside an can mediat e

spe ific antib dy me hanisms Thecomplement y st em can, howeve , also be brought int o action b the

surfac ()of mat erials for ign t o the b dy, inclu ing blo d-contacting medical devic s

[1 9][1 8]

The sy st em consis s of a n mbe of prot eins fou d in blo d that normaly cir ulat e as inactive

pr cursors The nomenclatur for the complement prot eins is “C” folowed b a simple Ara ic n mbe

for thenativeprot ein, an if clea ed, a smal “a ” or “b” t o in icat e the fra ment In the pr senc of a low

level of sp ntaneously formed r active C b, the pr senc of a biomat erial can trigg er an amplf ication

r sp nse w hose en r sult is prod ction of inflammat ory mediat ors, e.g C a an cyt ot oxic prot ein

complex es, e.g membrane at ack complex (MAC), w hich can s imulat e an ar a of inflammat ory

r sp nses inclu ing w hit e blo d c l (WBC) chemota is, r active o y g en spe ies (ROS)prod ction an

cyt okine ex r s ion

[1 9][1 0]

S e Figur B.2

The ear n me ous prot eins an prot ein fra ment that make up the complement y st em an these can

be divided int o thr e dis inct activ tion p thway s: the clas ical complement pathwa , the alt ernative

complement pathwa an the man ose-bin ing le tin pathwa It is the alt ernative pathwa that is

mos r g rded as being afe t ed b an r active t o the pr senc of medical mat erials

A n mbe of comme cial ELISA as ay s ar a aia le t o as es the amou t of complement prot ein in

blo d As C a is an ubiq itous fra ment amplfied d ring activ tion, this complement prot ein is

conside ed a g ood gene al indic ator of complement activ tion In ad ition, a soluble form of the t erminal

MAC a br viat ed SC b-9 can also be as es ed b ELISA as a SC b-9 is g ene al y conside ed a mor

imp rtant marke r pr sentative of the f l ex tent o c omplement ac ti vation Elev t ed levels of any of

complement comp nent in icat e activ tion of the complement sy st em High surfac ar a devic s such

as haemodialy sis f ilt ers and cardio ulmonary b pas devic s ha e be n as ociat ed with high levels

of activ t ed complement comp nent

[1 9]–[1 8][143]

an this phenomenon has be n lnked t o activ t e

leukocyt esan leukocyt e seq es ration in the lu gs

[1 0][1 7]

Measur ment of complement fra ment has seve al disadv ntag es Firs , ELISA kit only as a

complement component in the fluid phase ( e um or plasma); they do not measur the complement

comp nent w hich ar activ t ed an ad e e t o the devic surfac Depen ing on the natur of the

devic mat erial, the e could be signif icant amount of activ at ed complement on the device /mat erial

surfac w hich is u det ect ed b comme cial ELISA kit S con , the e is spe ies-spe if icity for many

of the comme cial y-a aia le kit an high b selne levels ar o se ved in ty ical in v itro t es ing

Th s, a pro riat e controls ne d t o be inclu ed an comp r d The clas ical CH-5 method a pears

useful with h man, b vine, p r ine an ra bit se um Howeve , sensitivity of CH-5 for det ecting

complement activ tion folowing contact with mat erials/devic s is low, given the CH-5 t es measur s

r s idu l complement activity an mos oft en only a smal p rtion of complement y st em is activ t ed b

mat erials/devic s Anothe fu ctional method of measur ment of complement activ tion in v itro is the

g ene ation of complement C - or C -conve tase det ermined b subs rat e conve sion R efe enc s [1 ]

an [1 ] also ad r s complement activ tion An ex E pro ides furthe information on conside ations

for complement t es ing of medical mat erials and devic s S e B.4 for detai s on g ene al ELISA

methodolo y

Key

biomaterial/device surface

NOTE F actor that are shown in red are me sura le by commer ial y availa le as ay kits

Figure B.2 — Alternative complement pathway

B.4 Methodolog y considerations for testing of plasma factors specific to

c oag ulation, platelet and leucocyte acti vation and c omplement acti vation using

ELISA (or other simi ar) techniques

B.4.1 General

As es ment of co gulation, plat elet, blo d c l an complement activity has long r led on clnical

as ay s that measur plasma levels of key prot eins formed in activ tion cascades or r leased from

activ tion or damag e t o v rious c ls ty es He e, clnical la orat ories oft en r ly up n t es kit that

use common enzyme-l nked immu osorbent as a t ech iq e In a b sic s u y, w hich ma be eithe

in v i vo or in v itro, implementation wi l depen up n a ai a i ty of a pro riat e antib dies/t es kit t o

spe ies-spe if ic targ et prot ein epit opes Ad itionaly, blo d samples wi be r trieved u de defined

con itions an pr p r d an analy sed pe as a ins ructions Defined con itions ma inclu e blo d

ex osur time, antico gulation an antico gulant level, t empe atur , flow, haemat oc it an othe

fact ors Comp rison of r sult t o a pro riat e controls, such as neg tive controls (e.g b sel ne levels

ar s atis icaly-significantly an biolo icaly-signif icantly highe than controls ma in icat e a

mat erial/devic design that pr sent a highe level of co gulation-, plat elet- or complement-mediat ed

risk t o the p tient Ex mple co gulation prot eins for w hich ELISA kit ar comme cial y a aia le

inclu e TAT (hrombin-antithrombin complex es), F 2 (fra ment r leased from prothrombin upon

formation of thrombin) and FPA ( fibrino eptide A r leased from f ibrinog en up n formation of f ibrin)

Simiarly, comme cial ELISA kit ar a ai a le for as es ing plat elet activ tion (e.g alpha gran le

r lease of BT and PF4 ) an complement activ tion (e.g C a an SC5 9 formation)

B.4.2 General as ay methods and documentation

B.4.2.1 General

Inclu ethe folowing with any t es r p rt

B.4.2.1.1 Referenc e documents

Man factur r ELISA prot ocol IFU in kit

B.4.2.2 Storag e and stabiity

Desc ibe al st orag e an s a i ity con itions of the kit r ag ent an the blo d/ lo d plasma being used

B.4.2.3 Proc ed r e

B.4.2.3.1 S mple pr eparation

Pro ide g ene al ins ructions

R esult of such t es ing ar inv ria ly depen ent up n surfac ar a ( SA) of a devic or devic t es

mat erial being t est ed F or this r ason, theSA-t o-blo d-v lume ratio (ex osur ratio) should be spe if ied

in each s u y Ex osur ratios of 3,0cm

2

t o 6,0 cm

2

/ml blo d (b sed on devic thick es ) ar consist ent

with ISO 1 99 -1 Othe ex osur ratios such as 1,5 an 2,0 times this ratio ma be worth conside ing

as highe surfac ar aswi theor ticaly inc easethe sensitivity of the r sponse t o the t es surfac

It is important t o spe ify the means of q enching the r action folowing the incub tion pe iod, i.e l s

name an conc ntration( ) of the q enching ag ent (s)

B.4.2.3.2 Diutio factor (DF)

Pro ide detai s on diution fact ors used, diuent used, et c

WARNING — Co siderable inter and intra do or-to-do or variabiity can be observed, making

a single ide l DF dif icult to identify A minimum of two test sample di utio factor is therefore

recommended to capture al sample values on the standard curve The sample absorbances

shal be in the rang e defined b the lowest and the highest standards If not, the samples sh uld

be retested with a new DF so that results fal within the rang e of the standard curve

B.4.2.3.3 Data selection in cases of testing with multiple DFs

If al or some samples ar t est ed un e con itions of no diution, low diution an high diution, r p rt

the v lues r q iring the smales DF that ar on the s an ard curve It is also ac epta le that if the “ no

diution” samples contain some of-the-curve v lues an al “low di ution” samples ar on the curve an

the “high di ution” samples contain some below-the-curve v lues, t o simply r p rt the “low diution”

r adingsw he e al v luesar on the curve u de the same DF

B.4.2.3.4 Preparation of standards

Desc ibe in detai how the s an ard curve an controlsar pr par d

B.4.2.3.5 Metho

Desc ibe in detai the o e al methods in theorde folowed, inclu ing highlghting any deviations from

the ELISA kit ins ructions

B.4.2.3.6 E aluation

Desc ibe in detai the calculations made t o det ermine the plasma levels of the prot ein being measur d

B.4.2.3.7 Statistical anal ysis

Desc ibe in detai the methods of s atis ical analy sis

B.4.2.3.8 Limitations and inter ferenc es

Lis al lmitations or int erfe ing fact ors such as incor e t blo d cole tion t ech iq e, e.g inadeq at e

mixing of the sample an citrat e solution (or othe antico gulant such a hep rin) ma lead t o falsely

elev t ed co gulation prot ein v lues; a wrong antico gulant ma lead t o elev tion of al b ckgrou d

v lues, et c

B.4.2.3.9 Referenc e inter val

Lis k own normal an a normal plasma levels of the prot ein being measur d an ex e t ed v lues of

controls

Inclu e sugg est ed diution fact or information, ELISA as a data she t forms, che kl s s for the ELISA

proc d r , et c

A nne x C

C.1 General considerations

Nume ous a pro ches ha e be n a pled t o as es the proc s an event of thromb sis Howeve ,

for blo d-contacting medical devic s an mat erials, the in v i vo natur of thromb sis has led t o the

methods desc ibed in C.2 an C.3 being mor commonly used t o as es devic -as ociat ed thromb sis

These methods ar used in ev luation of pe manent- an t emp rary-contact devic s As point ed out in

this document, the dive sity of blo d-contacting medical devic a plcations dictat es that in v i vo t es

models wi l be ne es ariy dive se, in orde t o a pro riat ely mimic each clnical a plcation

The method in C.2isac ept ed t o be the mos r lev nt manne in w hich t o as es devic s for thromb sis,

as he e theactual devic is ev luat ed in it int en ed clnical implant conf iguration in an animal model

The main element in this work ar algnment of the animal prot ocol ac ording t o ISO 1 9 3- 2, ac urat e

simulation of the h man clnical a plcation an inclusion in the prot ocol of a detai ed method( ) an

analyses t o be used t o as es the degr e of thrombosis

The method in C.3 is used les commonly an is not widely ac ept ed throughout the world Howeve ,

it ma be r q ir d or r q est ed b c rtain r gulat ory authorities The method has be n r fe r d t o

as the non-antico gulat ed venous implant (NA I) model (w hen antico gulation is not used) an the

antico gulat ed venous implant (A I) model (w hen antico gulation is inclu ed) The method it elf

inv lves inse tion of cathet er-sha ed devic s, or devic mat erials formed int o cathet er sha es,

int o the veins of animals for up t o 4 h folowed b gros as es ment of amou t of thrombus on the

mat erial/cathet er surfac The ca eat with the methodolo y ar shown in Ta le C.3 along with not ed

adv ntag es pro ided in Ta le C.4 Be a use of these ca eat , an t o a oid the false la el ng of mat erials

an devic s as thromb g enic in natur , data g ene at ed using this model r q ir s extr me caution in

int erpr tation

[143]

S e also A.3

When the t es devic is a cathet er-ty e devic used in a venous a plcation, the methods in C.2 an C.3

ar eq iv lent

C.2 In viv implant stud y of final devic e in pre-cl nical animal stud y

As s at ed in this document, in v i vo t es ing should be pe formed on devic s int en ed for in v i vo implant

a plcations ( e 6.1.6, 6.3.2 an 6.3.3) Prot ocols should inclu e detaied methods for a pro riat e

as es ment of blo d/devic int eractions, e.g analy seson

a) thedevic it elf,

b) blo d samples from the t es subje t, an

c) susc ptible tis ues an en org ns

Such pr clnical t es ing should use models that simulat e actual in-use a pl cation con itions, e.g

same implant sit e, g eometry, flow, contact d ration, t empe atur , st eri ty, et c ( e 6.1.2), an inclu e

a pro riat e trac a le controls, e.g a pr dicat e devic ( e 6.1.3) Importantly, t es ing should only

be done on actual complet e (f inished) devic s or component ( e 6.1.4), as t es ing on non-finished

prod ct an t es ing in p orly simulat ed use con itions wi not be highly pr dictive of pe formanc in

clnical a plcations ( e 6.1.6)

A ccordingly, devic s with ex v i vo a plcation an devic s with in v ivo a plcation should be t est ed in