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DOCKET SO. 8 4 s - 0 1 8 1

DRAFT

RECOMMENDED METHODS FOR

BLOOD GROUPING REAGENTS EVALLXTIOS

For further information abouc this draft. contact:

Center for Biologics Evaluation and Research H F B - 9 4 0 )

Food and Drug Administration

8 8 0 0

Rockville Pike

Bethesda. :4D 2 0 8 9 2

3 0 1 - 2 2 7 - 6 4 8 7 .

Submit vritten comments on this draft to:

Dockets Hanagement Branch H F A - 3 0 5 )


Rm 1 - 2 3

1 2 4 2 0 Parklawn Drive

Rockville. YD 2 0 8 5 7 .

Submit requests for single copies of t:his draft to:

Congressional. Consumer. and International

£

fairs Branch HFB-1 4 2 )


5 6 0 0 Fishers Lane

Rockville. YD 2 0 8 5 7

3 0 1 - 2 9 5 - 8 2 2 8

FAX

3 0 1 - 2 9 5 - 8 2 6 6 .

except that vritten requests delivered by carriers other than the U S

Postal Service for single copies of this draft should

be

submitted to:

Congressional. Consumer. and International ~f f a i r sStaff H F B - 1 4 2 )

Food and Drug -4dminis~ration

Suite 1 0 9 . Yletro Park Sorth 3

7 5 6 4 Standish Place

Rochill e. YD 2 0 8 5 5 .

Comments and requests should be identified with the docket number found in

brackets in the heading of this document.

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D R A F T

P R O P O S E D R E V I S I O N

RECOMMENDED METHODS FOR

BLOOD G R O U P I N G R E A G E N T S E V A L U A T IO N

MARCH 1 9 9 2

TABLE

O F C O N TE N TS

SUMMARY

COMMENTS AND CHANGES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PROPOSED RECOMMENDED METHODS

ABO BLOOD GROUP ING REAGENTS

R E F E R E N C E P R E P A R A T I O N S .............................1

P O T E N C Y T E S T I N G .................................... 2

S P E C I F I C I T Y T E S T I N G ................................

A V I D IT Y T E S T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T E S T F O R S P O N T A N E O U S A G G L U T I N A T I O N . . 11. - . - . - - - - . . . -

S L I D E AND M O D I F I E D T U B E R H BL OO D G R O U P I N G R E A G E N T S

R E F E R E N C E P R E P A R A T I O N S . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 2

P O T E N C Y T E S T I N G . . . .

...............................

1 3

S P E C I F I C I T Y T E S T I N G . . . . . . . . . . . . . . . . . . .............

16

A V I D I TY T E S T . .

....................................

2 2

T E S T F O R P R O ZO N E ..................................2 3

'I'EST FOR PRO ZO NE - METHOD 2 . . 2 5

LOW P ROTEIN RH BLOOD GROUP ING REAGENTS

R E F E R E N C E P R E P A R A T I O N S ............................2 7

P O T E N C Y T E S T I N G . . . . . . . . . . . . . . .....................2 8

S P E C I F I C I T Y T E S T I N G

...............................

3 2

A V I D I T Y T E S T . . . . , . . . . .............................

38

T E S T F O R S P O N T A N E O U S A G G L U T I N A T I O N

................

40

T E S T F O R P R O ZO N E

..................................

40

T E S T F OR P ROZONE METHOD 2..............-....-...42

R A R E B L O O D G R O U P I N G R E A G E N T S

R E F E R E N C E P R E P A R A T I O N S . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

P O T E N C Y T E S T I N G ...................................4

S P E C I F I C I T Y T E S T I N G ...............................4 8

A V I D I T Y T E S T ......................................3

i

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DR FT

ABOBLOODGROUPINGREAGENTS

Theserecommendedmethodsareprovidedtohelpassistmanufacturers

in pursuing new product licenseapplications and amendmentsto

existing product license applications. The methods,described

hereindonotbindtheagency,andmanufacturersmayconsideruse

ofothermethods. Incaseswheremanufacturerswishtousemethods

otherthanthosedescribedherein,FDArecommendsthatthematter

be discussed with FDA in advance. The methods, potency titer

values,specificityresults,andothermattersreferredtointhis

document are intended to assist manufacturers in preparing

submissionstoFDA. Theinformationisbasedoncurrentknowledge

and isnotmeanttobeallinclusiveandshouldnotbeviewedas

ensuring approvalor theonly means of achieving FDA approval.

Followingthemethodsprovidedinthisdocumentwillassistinthe

approvalprocess,butdoesnotguaranteeapproval. FDAwillreview

applicationsonanindividualbasisandapprovalswillbegranted

whensupportedby scientificevidence.

I. REFERENCEPREPARATIONS

A. TheReferenceBloodGroupingReagentslistedbelowcanbe

obtainedfrom:

CenterforBiologicsEvaluationandResearch

FoodandDrugAdministration

8800

RockvillePike

Bethesda,MD.

20892

USA

NOTE:

FDA Reference Blood Grouping Reagents are not

routinelyavailabletoanyoneexceptU.S. licensed

manufacturers and amounts issued will be

proportionaltolotsreleasedinthepreviousyear.

B. Reference sera are to be used according to the

accompanying package insert

for determining the

potencyofBlood~roupingeagentsaspartoftheirfinal

lotreleasetesting.

In-housereferencematerialsshouldbedevelopedforall

stability testing, in process testing or product

developmentpurposes.

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DRAFT

11. POTENCYTESTING

A. REAGENTDILUTIONS

1.

Beginning with the undildted reagent, prepare

separatemastertwo-foldserialdilutions(1in2,

1in 4,etc.

ofthetestreagentusing isotonic

saline containing1-2%bovine albumin-or=nother

diluent approved by the Director, Center for

Biologics Evaluation and Research. Test tubes

should be of a size that facilitates adequate

mixingofthecontents(12

X

75 mm

orlarger).

If the endpoint is expected to exceed 1024,

accuracywill be improved if direct intermediate

dilutionsaredone to keep the number of serial

transferstolessthan10. (e.g., Iftheexpected

endpointis4096,prepareaninitial1:10 dilution

withthesamediluentasusedabove.)

NOTE

:

All titrations should be carried to a

negativeendpoint. (SeeE.4)

2.

Prepare master dilutions of the Reference Blood

Grouping Reagent(s) as in paragraph 1 of this

section. For Anti-A,Band Anti-A and B prepare

dilutionsofeachReferenceBloodGroupingReagent

separately.

3 .

Aseparate,cleanpipetorpipettipshouldbeused

for each dilution (including any intermediate

dilutions) to avoid carryover of higher reagent

concentrations.

4.

Thelasttubeshouldcontaindiluentonlyandserve

as

diluentcontrol.

B.

REDBLOODCELLPREPARATIONS

Freshorfrozenredbloodcellsmaybeusedforpreparing

cellsuspensionsforthepotency testingof all Blood

GroupingReagentsunderthefollowingconditions:

1. Red blood cellsof any agemay be used,provided

the titer values of the reference reagents are

withinanacceptablerange.

2.

Redbloodcells

may

befrozenandthawedforusein

the preparation of cell suspensions for.reagent

evaluation. To ensurethatthecorrectcellhas

beenthawed,

appropriatecontrolsshouldbeusedto

demonstratethedesiredreactivityandidentityof

thethawedredbloodcellsonthedayofuse.

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DR FT

Themethodof freezing,storing,andthawingred

blood cells, including a description of the

cryoprotectivemediumshouldbedescribedindetail

andshouldbkapprovedbytheDirector,Centerfor

Biologics valuation and Researchbefore use in

controltestingoflicensedreagents.

3. Redbloodcellsshouldbewashedatleas~=twicen

isotonic sa$ine or until a clear supernate is

obtainedandthenresuspendedtoa2%suspensionin

isotonicsallineontaining1-2%bovinealbuminor

anotherdilqentapprovedby theDirector,Center

forBiologicbEvaluationandResearch.

C. MINIMUMTESTCELL6FORPOTENCY

REAGENT REDBLOODCELLS

Anti-A A, and

3

DIFFERENTA,B

Anti-B BandA,B

Anti-A, A,,A,

,

andB

Anti-AandB A,,A,

,

andB

ABcellswhichdonotreactwithanti-A,anddo

reactwithahti-H.

Acellsdhichdonotreactwithanti-A,anddo

reactwithahti-H.

D. THETEST(BYTUBEMETHOD)

1. Place0.1milliliterofeachreagentdilutionina

separate,cl$antesttube(approximately10

X

7 5 mm

or12

X

7 5 m r h .

2. Place 0.1 ailliliter of each Reference Blood

GroupingReagentdilutioninaseparate,cleantest

tube

(approx+mately10

7 5 u

or12

7 5

mm).

3. Add 0 1 milliliter of the appropriate

2%

cell

suspensiontbeachtesttube.

4. Mixthecontqntsofeachtubethoroughly. Incubate

for

5

minute$atroomtemperature(RT;20-30C)and

centrifugefor1 minuteatapproximately1000rpm

(100-125rcf)or15secondsatapproximately3400

rpm (900-lob0 rcf) or at a time and speed.

appropriateforthecentrifugebeingused.

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DRAFT

111,SPECIFICITYTESTING

A. REAGENTDILUTIONS

1.

Nodilutionofthereagentundertestispermitted.

B. REDBLOODCELLPREPARATIONS

Freshorfrozenredbloodcellsmaybeusedf~r'~re~aring

cellsuspensionsforthespecificitytestingofallBlood


1. Anycellsofanyagemaybeusedinthe"Testto

Confirm Reactivitywith Antigen Positive Cells1@

(IIIC.1). Inthe "Test to ConfirmAbsence of

contaminating Antibodiesw (III.C.2) licensed

reagentredbloodcellsmaybeusedanytimebefore

theirexpiration date. All otherredblood cell

samplesshouldbeusedwithin daysofcollection

fromthedonor.

Manufacturersthatwishtousecellsmorethan7

daysaftercollectionfromthedonoraretoobtain

approvalfromtheDirector,CenterforBiologics

Evaluation and Research, and are to provide

sufficientdatatosupporttherequest.

2. Redbloodcellsmeetingthecriteriaofparagraph1

ofthissectionmaybefrozenandthawedforusein

the preparationof cellsuspensionsforreagent

evaluation. Toensurethatthecorrectcellhas

beenthawed,appropriatecontrolsshouldbeusedto


thethawedredbloodcellsonthedayofuse. In

the ,case of cells expressing low frequency

antigens,testingforseveralcommonantigensmay

servetoadequatelyidentifythecell.

Themethodoffreezing,storing,andthawingred



andshouldbe

approvedbytheDirector,Centerfor

Biologics Evaluation and Research before use in


Licensedreagentredblood cellsmay beused as

provided.

Allotherredbloodcellsamplesshouldbewashed

atleasttwiceinisotonicsalineoruntilaclear

supernateisobtainedandthenresuspendedwithan

approveddiluenttothecellconcentrationlisted

inthemanufacturer's packageinsert.

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RAFT

-

.

C. MINIMUMTESTINGFORSPECIFICITY

1.

TESTTOCONFIRMREACTIVITYWITHANTIGENPOSITIVE

CELLS

a. At least4differentdonorswhoseredblood

cellsexhibitexpressionoftheantigenshould

betested.

b. When testing Anti-A,B and Anti-A and

B

reagents,thereactivitywithbothgroupAand

groupBredbloodcellsshouldbe confirmed

separately,i,e,atleastfourgroupAdonors

shouldbeused toconfirmthereactivityof

theAnti-AcomponentandatleastfourgroupB

donors should be used to confirm the

reactivityofthe

~nti-Bomponent.

c. Minimumtestredbloodcellsrecommended:


Anti-A A,(1)andA,B

(3)

Anti-B A,B (3)andB(1)

Anti-A,B A,(21, A,

(21,B(411

atleast3differentA,

Anti-Aand

B

A,(2), A,

(2),

B

(4),

atleast

3

differentA,

ABcellswhichdonotreactwithanti-A,and

doreactwithanti-H.

Acellswhichdonotreactwithanti-A,and

doreactwithanti-H.

A,cellsarerecommended;labelingshould

indicate detection of group A variants.

Include examples of I1strong

cellsIfand

I1moderate

cells1'

"Weak

cellsIfare

optional.

d. Includeatleastoneredbloodcellwhichdoes

notexhibitexpressionoftheantigenas a

negativecontrol.

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2 .

TESTTOCONFIRM-BSENCEOFCONTAMINATINGANTIBODIES

Testthereagentforthepresenceof antibodies

correspondingtothefollowingantigensbyoneof

themethodslistedbelow.

A B,

H

Lea,Leb,I,K,k,Kp",Kpb,Jsb,PI,D,C,

E, c,e,Cw,

N,S,s,U,Lu",Lub,Jkslkb,Fyal

F Y ~

Xg",DO",Dob,Yt",YtbILan,Co",Cob;Mg, Wrar

Sd",andVw.

If thesourcematerial isamonoclonal antibody

fromapreviouslycharacterizedandlicensedclone,

thislistmaybeshortenedasfollows:

A,B,

H

Lea,eb,I,K,k,Kpb,

Jsb,

PI,,C,E,

c ,

e,

M,

N,S,s,U,Lub,Jkslkb,FY",andFyb.

Approval for the use of fewer antigens than

includedon thislistmay berequestedfrom the

Director, Center for ~iologicsEvaluation and

Research by a manufacturer at the time of

submissionofthefirstprotocol.

a, Perform a direct test for thepresence of

contaminatingantibodiesby usingredblood

cellsfromatleast4differentdonorswhose

cellslacktheantigencorrespondingtothe

reagentantibody.

b. When red blood cells lacking the antigen

correspondingto thereagentantibodyunder

testarenotavailable,thereagentantibody

maybeadsorbedtoexhaustionwithcellsofa

knownphenotype.

Theadsorbedserummaythenbetestedagainst

red blood cellswhich exhibit any antigens

whichwerenotpresentonthecellsusedfor

adsorption. Themethodsforadsorptionand

subsequenttestingshouldbeapprovedby the

Director,CenterforBiologicsEvaluationand

Research,

c. When direct tests are impractical, the


Research may approve procedures whereby

antibodiesmay bepresumptivelyexcluded by

testinganappropriatenumberofnon-reactive

redbloodcellsamplestoprovidestatistical

assurance of the absence of contaminating

antibodies.

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DR FT

d. Red blood cell samplesfromfourdifferent

donorsmay beusedtoconfirmpresumptively

the absenceof contaminatingantibodies to

antigenshavinganincidenceofgreaterthan

99% inthegeneralpopulationoftheUnited

Statesorthecountryinwhichitissold.

D.

THETESTS

1. Toconfirmreactivitywithantigenpositivecells,

eachlotofBloodGroupingReagentshouldbetested

and results interpreted by all test methods

described in the manufacturer's package insert.

Minimumparameters (dropsofreagent,incubation

time,centrifugation,etc.)shouldbefollowed.

2. To confirmabsenceof contaminatingantibodies,


andresultsinterpretedbythemostsensitivetest

method(s) describedinthemanufacturer's package

insert. Maximum parameters (drops of reagent,

incubationtime,centrifugation,etc.) shouldbe

followed.

E. SPECIFICITYRESULTS

1. No hemolysis or rouleaux formation should be

detected by any of the methods in the

manufacturer's packageinsert.

Red blood cells which exhibit the antigen

correspondingtothereagentantibodyshouldyield

atleasta

2+

reaction. Ifanyofthefoursamples

testedyieldslessthana

2

reaction,redblood

cellsfromfouradditionaldonorswhoexhibitthe

antigenshouldbetested. Thetestisconsidered

satisfactoryifnomorethanoneofeightredblood

cellsamplesyieldslessthana 2 reactionwith

thetestreagent.

Whentestingunusualphenotypes,othercriteriafor

reactivity may apply. For example, a larger

percentageofA,redbloodcellsmaynotyield

a

2+

reactionwithAnti-A,BandAnti-Aand

B

butshould

yieldaclearlypositivemacroscopicresult.

3.

Thenegativecontrolcell(s)instepII1.C.

1

should

yield a negative reaction by each test method

describedinthemanufacturer's packageinsert.

4.

Testswithredbloodcellswhichlacktheantigen

correspondingtothe reagentantibody and tests

with adsorbed reagent should be negative,thus

confirmingtheabsenceofsignificantcontaminating

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R FT

.d = =. 4

4. Testswithredbloodcellswhichlacktheantigen

corresponding tothe reagent antibody and tests

with adsorbed reagent should be negative, thus


antibodies directed at the antigens listed in

III.C.2

5.

Themanufacturershouldliston thel~t-~release

protocol and in the n~pecificPerformance

Characteristicsnsection of the package insert

thoseredbloodcellantigenslistedinIII.C.2for

whichnospecificitytestshavebeenperformed.

Ifdesired,thered bloodcellphenotypeof the

antibodydonor(s)mayalsobelistedaspresumptive

evidencethatantibodiestothosefactorsarenot

present.

6

Confirmation by the manufacturer of n0nspeci:fi.c

reactionsafteralotofBloodGroupingReagenthas

beenreleasedshouldbereportedpromptlyby the

manufacturertotheDirector,CenterforBiologics

EvaluationandResearch.

IV. AVIDITYTESTFORSLIDEREAGENTS

A. REAGENTDILUTIONS

1.

Preparea

1

in

2

dilutionofthereagentundertest

bymixingequalpartsofthereagentandABserum

whichisfreeofantibodiesoradiluentapproved

by theDirector,CenterforBiologicsEvaluation

andResearch.


1.

Redbloodcellsshouldbepreparedaccording.othe


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C. MINImBl TESTCELLSFORAVIDITY


Anti-A A,,andA,B

Anti-B BandA,B

Anti-A,B A,,A,

,

B,and

,.

Anti-AandB A,,A,

,

B,and

ABce1l.swhichdonotreactwithanti-A,anddo

reactwithanti-H.

Ace1l.swhichdonotreactwithanti-A,anddo

reactwithanti-H.

A,redbloodcellsarerecommendedonlyifthe

reagent is recommended for detection of weak

subgroupsofAbyslidetechnique.

D.

THE

TEST(BYSLIDEMETHOD)

1.

Thetest istobe performedwith bothundiluted

reagentandthedilutedreagentpreparedinstep

1V.A by the method recommended in the


E. INTERPRETATIONOFTHETEST

1 -

Testresultsareobservedandrecordedatonehalf

ofthemanufacturer's recommendedobservationtime

andattheendofthefullrecommendedobservation

time.

. AVIDITYTESTINGRESULTS

1.

Signsofagglutinationshouldbeobservedwithboth

theundilutedanddilutedreagentattheendofthe

firsthalfoftheobservationtime.

2- Clearmaciroscopicagglutinationshouldbeobserved

withboththeundilutedanddilutedreagentatthe

endoftheobservationtimeandshouldbereported

asgreaterthanorlessthan1

mmindiameter.

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DRAFT

V .

TESTFORSPONTANEOUSAGGLUTINATION

A.

REAGENTDILUTIONS

1.


B.

REDBLOODCELLPREPARATION

1.

Obtaincpositivegroup

redblood

cells -@ram

one

donor.

2.

CoattheredbloodcellsheavilywithanIgGanti-c

suchthata

3-4+

directantiglobulintest(DAT)is

achievedandpositivereactionsareobtainedwitha

high protein h control reagent, but negative

reactionsareobtainedwith

a

salinecontrol.

Theexactprocedureforcoatingtheredbloodcells

will depend onthespecificantibodychosenfor

coatinganditsstrength.

C.

THETEST

1. Test the coated cell sample according to the

manufactu:rer8sackageinsert.

D. INTERPRETATION

1.

BloodGroupingReagentsforuseby alowprotein

tube test method should not spontaneously

agglutinateimmunoglobulincoatedredbloodcells.

2.

In the event that'thereagent under test does

agglutinate the coated red blood cells, an

effective control test or a control reagent

adequate to prevent misinterpretation of blood

groupresultsshouldberecommendedorsupplied.

3. Ifacontroltestorreagentisrecommendedbythe

manufacturer, cellsforuseincontroltestingin

sections11,11,and IV should giveanegative

directantiglobulintest(DAT).

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DRAFT

7

SLIDE*-ANDMODIFIEDTU E R LQOD ,GRQ?PJKREAGENTS

GENE,FIALINFORMATION

d

Theserecommendedmethodsareprovidedtohelpassistmanufacturers

in pursuing newproduct licenseapplicationsandamendmentsto

existing product licenseapplications. The methods described

hereindonotbindtheagency,andmanufacturersmaycons.ideruse

ofothermethods. Incaseswheremanufacturerswishtouse'methods


be

discussedwith FDA in advance. Themethods,potency titer




andisnotmeanttobeallinclusiveandshouldnotbeviewedas

ensuring approvalortheonlymeansofachieving FDAapproval.



applicationsonanindividualbasisandapprovalswillbegranted

whensupportedbyscientificevidence.


A .

TheReferenceBloodGroupingReagentslistedbelowcanbe

obtainedfrom:



8800 RockvillePike

Bethesda,MD. 20892

USA

Anti-D forevaluationofIgGproducts

Anti-c forrapidtubetest

Anti-E forrapidtubetest

Anti-c forrapidtubetest

Anti-e forrapidtubetest

NOTE FDAReferenceBloodGroupingReagentsarenot

routinely available to anyone except U.S.

licensedmanufacturersandamountsissuedwill

be proportional to lots released in the

previousyear.

B. All referencesera are to be used according to the

accompanyingpackage insert on y fordetermining the

potencyofBloodGroupingReagentsaspartoftheir.inal

lotreleasetesting.

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DRAFT


stability testing, in process testing or product


11.

POTENCYTESTING

A. REAGENTDILUTIONS

.

1. Beginning with the undiluted reagent, prepare

separatemastertwo-folddilutions(1in2,1in4,

etc.

of the test reagent using 20-22% bovine

albumin or another diluent approved by the

Director, Center for ~iologicsEvaluation and

Research. Testtubes should be of asizethat

facilitatesadequatemixingofthecontents(12

75

mmorlarger).

f the endpoint is expected to exceed 1024,

.ccuracywillbe improvedifdirectintermediate

.ilutionsaredonetokeepthenumberof serial

transferstolessthan10.(e.g., Iftheexpected

endpointis4096,prepareaninitial1:10dilution


NOTE

:

All titrationsshould be carried toa


2. Preparemaster dilutions of theReferenceBlood

Grouping Reagent(s) as in paragraph 1 of this

section. For testreagentscontainingmultiple

antibodies(ex.~nti-CDE)ilutionsofeachofthe

corresponding Reference Blood Grouping Reagents

shouldbemadeseparately.

3 .

separate,cleanpipetorpipettipshouldbeused


dilutions)to avoid carryoverof higher reagent

concentrations.

4.


asadiluentcontrol.



cellsuspensionsforthepotencytestingofallBlood


1. Redbloodcellsofanyagemay beused,provided

thetitervaluesof theReferenceBloodGrouping

Reagent(s)arewithinanacceptablerange.

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DR FT

2. Redbloodcellsmaybefrozenandthawedforusein

thepreparationof cell suspensionsfor reagent



demonstratethedesiredreaztivityandidentityof

thethawedredbloodcellsonthedayofuse.

Themethodof freezing,storing,andthauingred



andshouldbeapprovedbytheDirector,Centerfor

Biologics

v lu tion

andResearch before use in


3. Eachbatchofredbloodcellsforuseincontrol

testingofreagentsrequiringindirectantiglobulin

technique should be tested for absence of

complement or immunoglobulin molecules (DAT

negative)onthedayofuse.

4.

Redbloodcellsshouldbewashedatleasttwicein

isotonic salineor until a clear supernate is

obtainedandthenr2suspendedtoa2%suspensionin

isotonicsalinecontaining1-2%bovinealbuminor

anotherdiluentapprovedby theDirector,Center

forBiologicsEvaluationandResearch.

C. MINIMUMTESTCELLSFORPOTENCY


Anti-D Dce

Ror1

dCce

(r'r)

Anti-E dcEe

(rl1r)

Anti-c DCcEe

R,R,

Anti-e dcEe

(rl1r)

Anti-CD dCceandDce

(r'r andRor)

Anti-DE DceanddcEe

(Rorndr1Ir)

Anti-CDE dCceandDceanddcEe

(rtrndR,,r andrVtr)

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1. Place0.1 milliliterofeachreagentdilutionina

separate,cleantesttube(approximately

10X

7 5 mm

or12

X

7 5 mm).

2.

Place

0.1

milliliter of each Reference Blood

GroupingReagentdilutioninaseparate,

cieantest

tube(approximately10

X

75mmor12

X

7 5 mm).

3. Add 0.1 milliliter of the appropriate 2% cell

suspensiontoeachtesttube.

4. Mix the contents of each tube thoroughly and

incubatethetesttubesfor15minutesat37C.

5. -Centrifugefor2minutesatapproximately1000rpm

(100-125

rcf)or

5

secondsatapproximately

3400

rpm

(900-1000

rcf) or at a time and speed

appropriateforthecentrifugebeingused.

E.

INTERPRETATIONOFTHETEST

1.

Thecellbuttonsofeachtesttubeshou1.degently

dislodgedandexaminedmacroscopically:

2.

Thereactionsshouldbegradedasfollows:

4+

Cellbuttonremainsinoneclump.

3 + Cellbuttondislodgesintoseveralclumps.

2+

Cellbuttondislodgesintomanysinallclumps

ofequalsize.

1+ Cellbuttondislodgesintofinelygranular,

butdefinite,smallclumps.

D Cellbuttondislodgesintofinegranules,but

notdefinitesmallclumps. Resultsshouldbe

recordedasdoubtful. Forpurposesofthis

paragraph,doubtfulreactionsaredeemedtobe

negative.

0

Negativereaction-cellbuttondislodgesint.0

novisibleclumps.

3.

Thepotencytitervalueisthereciprocalofthe

greatestreagentdilutionforwhichthereactionis

gradedatI+.

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DRAFT

--a &

4Th+-dilution -caused4by Wie addition

o - fie

rep';

blood cells should not be considered as

contributingtothedilutionofthereagent.

4. Testresultsshouldshowatleastonetubewithno

agglutination after the endpoint. The diluent

controltubeshouldbenegative.

F. POTENCYTITERVALUES . -

1.

SlideandModifiedTubeRhBloodGroupingReagents

shouldhaveanaveragepotencytitervalueatleast

equaltothatofthereferencereagent.

2. Products recommended for use in automated or

microplate systems without user dilution (as

supplied)shouldbesufficientlypotentthatatwo-

fold dilution prepared with an approved diluent

.willproduce thesamequalitativetestresultas

the undiluted product when tested in accordance

withthemanufacturer's packageinsert.

111. SPECIFICITYTESTING

A. REAGENTDILUTIONS

1. Nodilutionofthereagentundertestispermitted.

B.





Any cellsofany agemay be used inthe"Testto

Confirm Reactivity with Antigen Positive Cellsw

(III.C.l). In the "Test to Confirm Absence of

Contaminating Antibodies" (III.C.2) Licensed


theirexpirationdate. All otherred blood cell

samplesshouldbeusedwithin7daysofcollection

fromthedonor.

Manufacturersthatwishtousecellsmore than

7


approval fromthe Director,Center forBiologics



2.

Redbloodcellsmeetingthecriteriaofparagraph1


the preparation of cell suspensions for reagent



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-

DR FT

P

.+; f

e

i g r e

rxsmE

c



the case of cells expressing low frequency





cryoprotectivemediumshouldbedescribed'-ndetail


~iologicsvaluationand Research before use in


3. Eachbatchofredbloodcellsforuseincontrol





4.

Licensedreagentredbloodcellsmay be used as

provided.



supernatantisobtainedandthenresuspendedwith

an approved diluent to the cell concentration

listedinthemanufacturer's packageinsert.

C.

MINIMUMTESTINGFORSPECIFICITY

1.


CELLS

a. Atleast

4

differentdonorswhoseredblood

cellsexhibitweakorheterozygousexpression

oftheantigenshouldbetested.

b. When testing reagents containing multiple

antibodies,thereactivityofeachspecificity

should be confirmed separately by using

4

differentredbloodcellspossessingonlyone

of the antigens for each different

specificity.

ex. For Anti-CDE reagents, at least four

donors should be used to confirm the

reactivityoftheAnti-Ccomponent,atleast

fourdonors should be used to confirm the

reactivityoftheAnti-D component,and at

leastfourdonorsshouldbeusedtoconfirm

thereactivityoftheAnti-Ecomponent.

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-

DR FT

c. Includeatleastoneredbloodcellwhichdoes

notexhibittheexpressionoftheantigenasa

negativecontrol.

TESTTOCONFIRMABSENCEOFCONTAMINATINGANTIBODIES

Testthereagentforthepresenceofantibodies



.

.

A,B,H,Lea,Leb,I,K,k,Kpa,Kpb,JsbtP,,D,C,

E, c,e,Cv,M,N,S,s,U,Lua,Lub,Jkalkb,~ y ~

FybIXgaIDoa,Dob,YtalYtbILan,CoalCob,Mq,Wra,

andSda.

If thesourcematerial isa monoclonal antibody



A,B,H,Lea,Leb,I,K,k,Kpb,Jsb,P,,D,

C,

E, c,

e,M N,S,s,U,Lub,Jkalkb,Fya,andFyb.


includedonthislistmay berequested fromthe

Director, Center for Biologics Evaluation and



a. Perform a direct test for the presence of


cellsfromatleast4differentdonorswhose


reagentantibody.


correspondingtothereagentantibodyunder

.testarenotavailable,thereagentantibody


knownphenotype.

Theadsorbedserumnaythenbetestedagainst

red blood ce'llswhich exhibit any antigens


adsorption. Themethods foradsorptionand

subsequenttestingshouldbeapprovedbythe

Director,

CenterforBiologicsEvaluationand

Research.


Director,CenterforBiologics

v lu tion

and


antibodiesmay bepresumptively excludedby



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DRAFT


antibodies.

d. Red blood cellsamplesfrom fourdifferent

donorsmaybeused toconfirmpresumptively

the absence of contaminating antibodiesto


99%

inthegeneralpopulationoftheUnited

States.

,

3. TESTTOCONFIRMABSENCEOFANTI-AANDANTI-B

a. GroupA,and B redblood cells lackingthe

antigencorrespondingtothereagentantibody

shouldbetested. GroupA,Bredbloodcells

maybesubstitutedforA,and/or Bredblood

cellsifeitherareunavailable.

Adsorbedserummay be used as inIII.C.2.b

above.

4.

PHENOTYPESRECOMMENDEDFORTESTING

As a minimum, red blood cells exhibiting the

following phenotypes should be used in the

specificitytestingoutlinedinsteps1,2,and

3

above.

MINIMUM REDBLOODCELLS

DCce,Dce,dCce,anddcEe

(R,r,Ror,rtr,ndrMr)

A,dce,Bdce,and dce(rr)

Vwpositive

3 differentdce(rr)Bg(a+)

*

6Dusamplesrepresentingdifferent

Rhphenotypesand reactiveby the

IndirectAntiglobulinTestonly

Anti-D CategoryIV,V,andVIcells

(monoclonal)

Dce,dCce,anddcEeordcE

(Rr,rtr,ndrI1rorr"r'l)

C+Ce-(e..R,R,or,R,r)

* *

Cwpositive(e.g.RlUr)

A,dce,Bdce,and

dce(rr)

Dce,dCceordCe,anddcEe

(Rr,rtrorrtrt andrV1r)

E+cE-(e.g.R,R,orR,r)

A,dce,B dce,and

dce(rr)

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DRAFT

E . SPECIFICITYRESULTS

1.

No hemolysis or rouleaux formation should be

detected by

any of the methods in the


2. Red blood cells which exhibit the antigen


atleasta2+reaction. Ifanyofthefour'samples

testedyieldslessthana2+reaction,redblood




cellsamplesyieldslessthana2 reactionwith

thetestreagent.

Whentestingunusualphenotypes,

othercriteriafor

reactivity may apply. For example, a larger

percentageofC+Ce-redbloodcellsmaynotyield

a 2+ reaction with Anti-C but should yield a

clearlypositivemacroscopicresult.

3.

T h e n e g a t i v e c o n t r o l c e l l s ) instepIII.C.1should




correspondingtothereagentantibody and tests




5.

Themanufacturer should listonthe lotrelease

protocol and in the "specific Performance

Characteristics" section of the package insert

thoseredbloodcellantigenslistedinI11.C.2for


Ifdesired,theredbloodcellphenotypeof the



present.

6 .

TestswithgroupA,andBredbloodcellsshouldbe

negative,thusconfirmingtheabsenceofanti-Aand

anti-B.

7. Confirmation by the manufacturer of nonspecific

reactionsafteralotofBlood~roupingeagenthas


manufacturertothe~irector,enterforBiologics


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DRAFT


A. REAGENTDILUTIONS

1.

Preparea1 in2dilutionofthereagentundertest

bymixingequalpartsofthereagentandABserum,

groupcompatibleserum,oradiluentapprovedby

theDirector,CenterforBiologics~valuatfonnd

Research.

1.

Redbloodcellsshouldbepreparedaccordingtothe


C. MINIMUMTESTCELLSFORAVIDITY


Anti-D DCe(R,r)

dCce(rtr)

C+Ce-(R,R,orR,r)

Cwpositive(R,'r)

Anti-E dcEe(rVVr)

E+cE-(R,R,orR,r)

Anti-c dCce(rtr)

Anti-e dcEe(rHr)

Anti-CD DceanddCce

(R,randrtr)

rGrorrVVCr

Anti-DE DceanddcEe

(RrandrNr)

Anti-CDE Dce,dCce,anddcEe

(Ror,tr,ndr"r)

rCr

*

*

Only if the reagent is recommended for

detectionoftheGantigenbyslidetechnique.

D. THETEST(BYSLIDEMETHOD)

1. Thetestistobe performedwith both undiluted

reagentandthedilutedreagentpreparedinstep

IVtA by the method recommended in the


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DRAFT

E .

INTERPRETATIONOFTHETEST

1.

Testresultsareobservedandrecordedatonehalf



time.

F. AVIDITYTESTINGRESULTS

1.


theundilutedanddiluted reagentatonehalf of

therecommendedobservationtime.

2. Clearmacroscopicagglutinationshouldbeobserved


endoftherecommendedobservationtimeandshould

bereportedasgreaterthanorlessthan

1

mm.

V. TESTFORPROZONE

A . REAGENTDILUTIONS


B.


1. Obtain at least three red blood cell samples

representingthreedifferent

Rh.

phenotypes which

exhibit heterozygous or weak expression of the

antigencorrespondingwiththereagentantibody.

2. Freshorfrozenredbloodcellsmaybeused under

thefollowingconditions:

a. Licensedreagentredbloodcellsmay beused

an.ytimebeforetheirexpirationdate.

b. Frozenredbloodcellsshouldhavebeenfrozen

within

daysofcollectionfromthedonorand

shouldbeusedonthedayofthawing.

c. All other red blood cell samplesshould be

used within days of collection from the

donor.

3. Red blood cells should not be coated with

complement or immunoglobulin (should be direct

antiglobulintestnegative).

4.

Licensed reagent red blood cellsmay be used as

provided.



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. DRAFT




C.

CELLSSUGGESTEDFORUSE

N

THETESTFORPROZONE

.


DCce

(R,r

1

DCcEe

RlR2

~nti-E DCcEe

RlR2

1

DCceandDCcEe

(R,randRlR2)

DcEeandDCcEe

(R,randR,R,)

D.

THETEST

1.

For each cell sample tobe tested label three

tubes,

"15

minutestt,tt30minutestt and

6

minutestt

respectively.

2.

Add the appropriateamountof thereagentunder

testtoalltubes.

Ifthemanufacturer's packageinsertrecommendsthe

useof

* l

dropofreagent,use

1

dropforthistest.

Ifthemanufacturer'spackageinsertrecommendsthe

use of

2

dropsof reagentor

1

or

2

dropsof

reagent,use

2

dropsforthistest.

3.

Add

1

dropofeachcellsampletoitsrespective

tubes.

4.

Mixandincubateforthetimeindicatedonthetube

label according to the manufacturer's package

insert,i.e. atthetemperaturerecommended for

thosetestsgivinganegativeresult.

5.

Centrifuge according to the package insert and

examineforagglutination. Gradethereactionsas

in

1I.E.

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DRAFT

E, INTERPRETATIONOFTHETEST

1,

Ifthereactiongradesarethesgmeorincreaseas

the incubation time increases, no prozone is

present.

2. Ifthereactiongradesdecreaseastheincubation

timeincreases,aprozoneispresent.

F. RESULTS

1.

Atleasta2+reactionshouldbeobtainedwithALL

samplesatALLincubationtimes.

VI. TESTTODETECTPROZONES

METHOD

2

A.

REAGENTDILUTIONS

1.

Preparea1+5dilutionofthereagentundertestin

inerthumanserum(groupABorcompatiblewiththe

cellstobetested).


1. SeeV.B.

C.

CELLSSUGGESTEDFORUSEINTHETESTFORPROZONE

1. SeeV.C.

D. TH.ETEST

The1+5dilutionandundilutedreagentwillbetestedin

parallel.

1. For each cell to be tested,label twosets of

tubes, nI.S.w, "1 minuten, " 3 minutes", "5

minutesu,and"10minutes".

2.

Add theappropriateamountof thereagentunder

testtoalltubes.

Ifthemanufacturerfspackageinsertrecommendsthe

useof1dropofreagent,use1dropforthistest.

Ifthemanufacturerfspackageinsertrecommendsthe

use of

2

dropsof reagentor 1 or

2

drops of

reagent,use2dropsforthistest.

3.

Add

1

dropofeachcellsampletoitsrespective

tubes.

4.

Mixandincubateatroomtemperatureforthetime

indicatedonthetubelabel.

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--

. .

DR FT

LOWPROTEIN

R

BLOODGROUPINGREAGENTS

GENERALINFORMATION

Theserecommendedmethodsareprovidedtohelpassistmanufac:turers

in pursuing newproduct licenseapplicationsandamendmentsto

existing product licenseapplications. The methods described

hereindonotbindtheagency,andmanufacturersmayconsitleruse



be discussedwith FDA inadvance. Themethods,potencytiter





ensuringapprovalortheonlymeansof achievingFDAapproval.

Followingthemethodsprovidedinthisdocument

willassistinthe


applicationsonanindividualbasisandapprovalswillbeqranted



A. TheReferenceBloodGroupingReagentslistedbelowcanbe

obtainedfrom:



8 8

RockvillePike

Bethesda,MD 2 892

USA

Anti-CDforevaluationofIgM,Anti-D

products

Anti-C forsalinetubetest

Anti-E forsalinetubetest

NOTE:

FDA Reference Blood Grouping Reagents are not

routinelyavailabletoanyoneexceptU.S. licensed

manufacturers and amounts issued will be

proportionaltolotsreleasedinthepreviousyear.

ForBloodGroupingReagentsforwhichthereisno

ReferenceBloodGroupingReagent,itisstrongly

recommended that a previously approved lot of

reagentbeusedasanin-housecontrolreaglent.

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B. All reference sera are to be used according to th,e

accompanying package insertonly fordetermining the

potencyofBloodGroupingReagentsaspartoftheirfinal

lotreleasetesting.


stability testing, in process testing or proauct


22 . POTENCYTESTING

A.

REAGENTDILUTIONS

1. Beginning with the undiluted reagent, prepare

separatemastertwo-foldserialdilutions(1in2,

1in4,etc.) ofthetestreagentusingisotonic

salinecontaining1-2%bovinealbuminoranother

diluent approved by the Direckor, Center for




X

75

orlarger).

If the endpoint is expected to exceed 1024,

accuracywill be improvedifdirectintermediate

dilutionsaredonetokeepthenumberof serial

transferstolessthan10. (e.g., Iftheexpected

endpointis4096,prepareaninitial1:10 dilution


NOTE: All titrations should becarried to a


2.

Preparemaster dilutions of the Reference Blood

GroupingReagent(s)

orin-housecontrolreagentas

inparagraph1ofthissection. Fortestreagents

containing multiple antibodies (ex. Anti-CDE),

dilutionsofeachofthecorrespondingReference

BloodGroupingReagentsshouldbemadeseparately.

3 .



dilutions) to avoid carryoverof higher reagent

concentrations.

4. Thelasttubeshouldcontaindiluentonlyandserve

asadiluentcontrol.

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Anti-D Dce

(

Ror

Anti-C dCce

(r'r)

Anti-E dcEe

(rttr

Anti-c DCcEe

(RIR2

Anti-e dcEe

(rltr)

Anti-CD dCceandDce

(r'r andRor

Anti-DE DceanddcEe

(Rorndrl1r)

Anti-CDE dCceandDceanddcEe

(rlrand&r andrwr)


1. Place0.1milliliterofeachreagentdilutionina

separate,cleantesttube(approximately10

7 5

mm

or

2 7 5

mm).

2.

IfaReferenceBloodGroupingReagentisavailable,

place 0.1 milliliter of each Reference Blood

GroupingReagentdilutioninaseparate,cleantest

tube(approximately1

7 5

rn or12

7 5

mm).

3. Add 0.1 milliliter of the appropriate 2% cell

suspensiontoeachtesttube.

4. Mix the contents of each tube thoroughly and

'incubatethetesttubesat3 7

Cfor15minutes.

If no Reference Blood Grouping Reagent is

available,incubateat

37

Cfortheshortestperiod

oftimerecommendedinthemanufacturer's package

insert.

5.

Centrifugefor1minuteatapproximately1000rpm

(100-125rcf)or15secondsatapproximately.3400

30

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DR FT

rpm (900-1000 rcf) or at a time and speed

appropriate for the centrifuge being used.

In the case of reagents for which no Reference

Blood Grouping Reagent is available, centrifuge for

the shortest period of time at the lowest speed

recommended in the manufacturer's package insert.

,

E .

INTERPRETATION OF THE TEST

1.

The cell buttons of each test tube should be gently

dislodged and examined macroscopically.

2.

The reactions should be graded as follows:

4+ Cell button remains in one clump.

3 +

Cell button dislodges into several clum]ps.

2+ Cell button dislodges into many small clumps

of equal size.

1+ Cell button dislodges into finely granular,

but definite, small clumps.

D

Cell button dislodges into fine granule:;, but

not definite small clumps. Results should be

recorded as doubtful. For purposes of' this

paragraph, doubtful reactions are deemed to be

negative.

0

Negative reaction- cell button dislodges into

no visible clumps.

3. The potency titer value is the reciprocal of the

greatest reagent dilution for which the reaction is

graded at l+.

The dilution, caused by the addition of the red

blood cells should not be considereld as

contributing to the dilution of the reagent.

4.

Test results should show at least one tube with no

agglutination after the endpoint. The diluent

control tube should be negative.

F.

POTENCY TITER VALUES

1.

Products for which Reference Blood Grouping

Reagents are available should have an average

potency titer value at least equal to that of the

reference reagent.

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2. Productsofpolyclonaloriginwhicharerecommencled

for tube test methods for which there are no

ReferenceBlood~roupingeagentsavailableshould

have

a

potency titer value of at least a

1+

reactionwitha1:4 dilutionofreagent:

eg.Anti-c (saline)

Anti-e (saline)

3.

Productsofmonoclonaloriginwhicharerecommendied

for tube test methods for which there are no

ReferenceBloodGroupingReagentsavailableshou,ld

have a potency titer value of at least a 1+

reactionwitha

1:8

dilutionofreagent:

eg.Anti-c(saline)

Anti-e(saline)

Manufacturersthatwishtoestablishpotencytiter

valuesotherthanthesearetoobtainapprovalfrom

theDirector,Centerfor~iologicsvaluationand

Researchatthetimeoflicenseapplication.

4.

Products recommended for use in automated or

microplate systems without user dilution as

supplied)shouldbesufficientlypotentthatatwo-

fold dilution prepared with an approved diluent

will produce thesamequalitativetestresult as

the undiluted product when tested in accordance


111.SPECIFICITYTESTING

A. REAGENTDILUTIONS






1.

Any cellsofanyagemaybe usedinthe '!Test to

Confirm Reactivity with Antigen Positive Cells"

(III.C.l). In the '!Test to Confirm Absence of

Contaminating Antibodiesgg (III.C.2) licensed


theirexpirationdate. All otherred blood cell

samplesshouldbeusedwithin

7

daysofcollection

fromthedonor.

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DR FT

Manufacturersthatwishtousecellsmorethan

7


approvalfromtheDirector,Center.forBiologics



2 .

Redbloodcellsmeetingthecriteriaofpaqagraph1

ofthissectionmaybefrozenandthawed$orusein

the preparationofcell suspensionsforreagent

evaluation. Toensurethatthecorrectce.11has

beenthawed,




the case of cells expressing low frelquency



Themethodoffreezing,storing,andthawingred




Biologics Evaluation and Researchbefore use in


3.

Eachbatchofredbloodcellsforuse'incontrol





4. Licensedreagentredbloodcellsmay beused as

provided.







1.

TEST

TO

CONFIRMRE CTIVITY WITH

NTIGEN

PO:~ITIVE

CELLS

a. Atleast

4

differentdonorswhoseredblood

cellsexhibitweakor

heterozygousexpression


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DR FT



should be confirmed separately by using 4

differentredbloodcellspossessingonlyone

of the antigens for each different

specificity.

ex. For Anti-CDE reagents,at least four

donors should be used to confirm .the

reactivityoftheAnti-Ccomponent,atleast

fourdonorsshould be used to confirm 'the

reactivityof theAnti-D component,and at

leastfourdonorsshouldbeusedtoconfirm

thereactivityoftheAnti-Ecomponent.



negativecontrol.

2.

TESTTOCONFIRMABSENCEOFCONTAMINATINGANTIBODIES

Testthereagentforthepresence of antibodies



A,BI

I

Lea,Leb,I,.K,k,Kpa,Kpb,Jsb,P,,

D,

C,

E, c,e,Cw,MIN,S,s,U,Lua,Lub,Jka,Jkb,E'ya,

Fyb,Xga,Doa,Dob,Yta,Ytb,Lan,CoatCob,M9,91ra,

andSda.

If thesourcematerial isamonoclonal antib'ody



A,

B

I Lea,eb,I,K k,Kpb,Jsb,I, C,E

C

e,

MIN,S,s,U,Lub,ka,Jkb,Fya,andFyb.


includedonthislistmay be requestedfromthe




a. Perform adirect test for thepresence of


cellsfromatleast

differentdonorswhose


reagentantibody.

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DRAFT





knownphenotype.

Theadsorbedserummaythen

betestedagainst

red blood cellswhich exhibitany.antigens



subsequenttestingshouldbeapprovedbythe

Director,CenterforBiologicsEvaluatiolnand

Research.




antibodiesmay be presumptivelyexcludedby




antibodies.

d. Red blood cell samplesfrom fourdifferent


the absenceof contaminating antibodies to


99

inthegeneralpopulationoftheUnited

States.

3. TESTTOCONFIRMABSENCEOFANTI-AANDANTI-B

a. GroupA, and B redblood cellslackingthe


shouldbetested. GroupA,Bredbloodcells

maybesubstitutedforA,and/orBredblood


Adsorbed serummay be usedas in III.C.2.b

above.

4.

PHENOTYPESRECOMMENDEDFORTESTING

As

a

minimum, red blood cells exhibiting the

following phenotypes should be used in the

specificitytestingoutlinedinsteps

1,

2,and 3

above.

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R E A G E N T

A n t i - D

A n t i - D

( m o n o c l o n a l )

R E D B L O OD ' C E L L S

D C c e , D c e , d C c e ,

a n d

d c E e

( R , r , R o r , r l r

and

r I 1 r )

A,

dce

B

dce and 0 dce

( r r )

Vw p o s i t i v e

d i f f e r e n t

dce

( r r ) B g ( a + )

c e l l s

*

6

Du

samples

r e p r e s e n t i n g

d i f f

ererlt

R h pheno types and

r e a c t i v e by

I n d i r e c t A n t i g l o b u l i n T e s t o n l y

*

C a t e g o r y I V ,

V ,

and V I

c e l l s

D c e , d C c e ,

and

d c E e

o r

d c E

( K r ,

r l r and r w ro r

r n r w )

C + C e - ( e x . R zR 2

o r

R , r )

* *

Cw p o s i t i v e ( e x . R l w r )

A, dce B dce and

0

dce ( r r )

D c e , d C c e

o r

d C e ,

and

d c E e

( K r ,

r l r o r

r l r l and r l l r )

E + c E - ( e x . R z R , o r R , r )

A,

dce

B

dce

a n d

0 dce

( r r )

d C c e

and

D C E e o r DCE o r d C E

( r l r and R,R,

o r

R,R,

o r

r Y r Y )

A , D C e ,

B

D C e ,

a n d 0

D C e ( R , R , )

D C c E e

f

neg ( R l R 2 )

d c E e

and

D C c E o r DCE o r d C E

( r u r

and

R,R2 o r R,R, o r r Y r Y )

A, D c E , B D c E ,

and 0

D c E ( R , R , )

D C c E e

f neg

( R l R 2 )

D c e , d C c e ,

and

d c E

o r

d c E e

( R , r , r l r

and

r u r w

o r

r W r )

A,

d c e

B

dce

and

0 dce

( r r )

rG ro r r I g G r

***

D c e , d C c e

o r

d C e ,

and

d c E e

( K r ,

r l r

o r

r l r l a n d

r U r )

A,

dce

B dce

and 0 dce

( r r )

D c e , d C c e , and d c E e

( K r ,

r l r

and r u r )

A,

dce

B

dce

a n d

0 dce ( r r )

rG r***

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DRAFT

*

ForAnti-DreagentsrecommendedforD"testing.

* * r'"r cellsmaybeusedinadditiontobutnot

asasubstituteforC+Ce-cells

* * *

Recommendediflabelingindicatesdetectionof

antigen

. . .

D. THETESTS

1.

Toconfirmreactivitywithantigenpositivecells,




Minimum parameters (dropsof reagent, inculbation

time,centrifugation,etc.)shouldbefollowced.

2. To confirm absence of contaminating antibodies,



method(s) describedinthemanufacturer's package

insert. Maximum parameters (dropsof reagent,

incubationtime,centrifugation,etc.) shou~ldbe

followed.





2. Red blood cells which exhibit the antigen


atleasta2+reaction. Ifanyofthefoursamples

testedyieldslessthana 2+reaction,redblood




cellsamplesyieldslessthana2+reactionwith

thetestreagent.

Whentestingunusualphenotypes,othercriteriafor

reactivity may apply. For example, a llarger

percentageofC+Ce-redbloodcellsmaynotyield

a 2+ reaction with Anti-C but should yield a

clearlypositivemacroscopicresult.

3. Thenegativecontrolcell(s)instep1II.C.1should



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4.

Testswithredbloodcellswhichlacktheantigen

correspondingto thereagent antibody and tests




III.C.2

5.

Themanufacturershould listonthe lotrelease

protocol and in the "Specific Performance

Characteristics" section of the package insert

thoseredbloodcellantigenslistedinI11

.C.

2for


Ifdesired,theredbloodcell phenotypeof the



present.

6.

TestswithgroupA,andBredbloodcellsshould:be

negative,thusconfirmingtheabsenceofanti-Aand

anti-B.

7.

Confirmation by the manufacturer of nonspecific

reactionsafteralotofBloodGroupingReagenthtas

beenreleasedshouldbereportedpromptlyby tlhe

manufacturertotheDirector,CenterforBiologilcs


AVIDITYTESTFORSLIDEREAGENTS

A.

REAGENTDILUTIONS

1.

Preparea

1

in2dilutionofthereagentundertest

bymixing,qualpartsofthereagentandABseruim,

groupcompatibleserum,oradiluentapprovedIby

theDirector,CenterforBiologicsEvaluationand

Research.


1.

Redbloodcellsshouldbepreparedaccordingtotlhe


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DRAFT



Anti-D DCe(R,r)

Anti-C dCce(rfr)

C+Ce-(R,R,orR,)

Cwpositive(RlWr)

dcEe(rttr)

E+CE-(R,R,orR,r

dCce(rfr)

dcEe(rttr)

DceanddCce

(R,randrfr)

rCr orrttCr

DceanddcEe

(Rrandrmr)

Dce,dCce,anddcEe

(Rorffrfndrttr)

rGr

*

*

Only if the reagent is recommended for

detectionoftheGantigenbyslidetechnique.


1. Thetest is tobe performedwith bothundiluted

reagentandthedilutedreagentpreparedin step

IV,A by the method recommended in the

manufacturerfspackageinsert.


1. Testresultsareobservedandrecordedatonehalf



time.

F. AVIDITYTESTINGRESULTS

1.


theundilutedanddilutedreagentatonehalfof

themanufacturerfsrecommendedobservationtime.



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endofthemanufacturer's recommendedobservati.on

timeandshouldbereportedasgreaterthanorless

than mm.

V. TESTFORSPONTANEOUSAGGLUTINATION

A. REAGENTDILUTIONS

..,


B. REDBLOODCELLPREPARATION

1.

Obtaincpositivegroup

redbloodcellsfromone

donor. (WhentestinganAnti-creagent,useRh(D)

positivegroup

cells.)

2.

CoattheredbloodcellsheavilywithanIgGanti-c

(anti-DwhentestinganAnti-creagent)suchthat.a

3-4+directantiglobulintest(DAT)isachievedand

positivereactionsareobtainedwithahighprotein

Rh control reagent, but negative reactionsare

obtainedwithasalinecontrol.

Theexactprocedureforcoatingthered.bloodcells

will dependonthespecificantibodychosenfor

coatinganditsstrength.

C.

THETEST

1. Test the coated cell sample according to the


D. INTERPRETATION

1. BloodGroupingReagentsforusebythesalinetube

testmethod shouldnotspontaneouslyagglutinate

immunoglobulincoatedredbloodcells.

2.

In the event that the reagent under test does

agglutinate the coated red blood cells, an

effective control test or a control reagent

adequate to prevent misinterpretation of blo~od

groupresultsshouldberecommendedorsupplied.

VI. TESTFORPROZONE

A. REAGENTDILUTIONS

1.


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D.

THE

TEST

1.

For each cell sample to be tested label three

tubes,"15minutestt,30minutestt,nd"60minutestt

respectively.

2. Add theappropriateamountof thereagentunder

testtoalltubes.

Ifthemanufacturer'spackageinsertrecommendsthe

useof1dropofreagent,use1dropforthistest.

Ifthemanufacturer's packageinsertrecommendsthe

use of 2 dropsof reagent or 1or

2

drops of

reagent,use2dropsforthistest.

3. Add 1dropof eachcellsampletoitsrespective

tubes.

4. ix

andincubateforthetimeindicatedonthetube

label according to the manufacturer's package

insert,i.e. at thetemperaturerecommended1for

thosetestsgivinganegativeresult.

5. Centrifuge according to the package insert.and

examineforagglutination. Gradethereactio:nss

in1I.E.

1. Ifthereactiongradesarethesameorincreaseas

the incubation time increases, no prozone is

present.

2. Ifthereactiongradesdecreaseastheincubation

timeincreases,aprozoneispresent.

F. RESULTS

1. Atleasta2+reactionshouldbeobtainedwithALL

samplesatALLincubationtimes.

VII.TESTTODETECTPROZONES

METHOD2

A. REAGENTDILUTIONS

1.

Preparea1+5dilutionofthereagentundertestin

inerthumanserum(groupABorcompatiblewiththe

cellstobetested.)


1.

SeeV1.B.

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I

DRAFT

RAREBLOODGROUPINGREAGENTS

GENERALINFORMATION

Theserecommendedmethodsareprovidedtohelpassistmanuf

aciturers

in pursuing new product licenseapplicationsand amendmentsto

existing product license applications. The methods described

hereindonotbindtheagency,andmanufacturersmayconsidleruse



e discussedwith FDA in advance. Themethods,potency titer

values,specificityresults,andothermattersreferredtoi.nthis


submissionstoFDA. Theinformationisbasedoncurrentknotwledge


ensuringapprovalortheonlymeansof achievingFDAapproval.



applicationsonanindividualbasisandapprovalswillbeqrranted



A. TherearenoReferenceBloodGroupingReagentsava.ilable

for the reagents covered in this doc:ument. It is

stronglyrecommendedthatapreviouslyapprovedlotof

reagentbeusedasanin-housecontrolreagent.

POTENCYTESTING

A. REAGENTDILUTIONS

1.

Beginning with the undiluted reagent, prepare

separatemastertwo-foldserialdilutions 1. in

2,

1 in4,etc.)ofthetestreagentusingisotonic

salinecontaining1-2%bovine albuminoramother

diluent approved by the Director, Center for




75

mm orlarger).

f

the endpoint is expected to exceed 1024,

accuracywill be improvedif directintern~ediate

dilutionsaredonetokeepthe number of serial

transferstolessthan10.(e.g., Iftheexpected

endpointis4096,prepareaninitial1:10dilution


NOTE : All titrationsshould be carried to a


2 Preparemasterdilutionsofthein-housecontrol

reaqentasinparagraph1ofthissection.

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DRAFT

3 .



dilutions)to avoidcarryoverof higher reaqent

concentrations.

4.


asadiluentcontrol.



cellsuspensionsforthepotencytestingof allBlood


1. Redbloodcellsofanyagemaybeused,provided

thetitervaluesofthein-housecontrolreagent

arewithinanacceptablerange.

Redbloodcellsmaybefrozenandthawedforusein

the preparation of cell suspensionsfor reaqent


beenthawed,




the case of cells expressing low.frequency







Biologics Evaluation and Research as a license

amendmentbeforeuseincontroltestingofreagent.

3.

Eachbatchofredbloodcellsforuseincontrol





4.

Redbloodcellsshouldbewashedatleasttwicein

isotonic saline or until a clear supernate is

obtainedandthenresuspendedtoa2%suspensionin

isotonicsalinecontaining1-2%

bovinealbuminor

anotherdiluentapprovedby theDirector,Center

forBiologicsEvaluationandResearch.


1. At least 2 different donors with phenotypes

exhibitingweakand/orheterozygousexpressionof

theantigen,whereapplicable.

Forexample,Anti-LeaandAnti-Lebareexcluded.

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DRAFT

3 .

The po te n cy

t i t e r

v a l u e i s t h e r ec ip ro ca l of t h e

g r e a t e s t r e a g e n t d i l u t i o n f o r w h i c h t h e r e a c t i o n i s

g rad ed a t l+.

The d i l u t i o n caused by t h e a dd i t i on o f t h e r e d

b l ood c e l l s s hou l d n o t be c o n s i d e r e d a s

c o n t r i b u t i n g t o t h e d i l u t i o n of t h e r ea ge n t .

4 . T e s t r e s u l t s s h ou ld show a t l e a s t o ne t u b e w i t h n o

a g g l u t i n a t i o n a f t e r t h e e ndp o in t . The d i l u e n t

c o n t r o l t u b e s h o u l d be n e g a t i v e .

F. POTENCY TITER VALUES

1.

Produ c t s o f po l y c l on a l o r i g i n whi ch a r e recomnlended

f o r t u b e t e s t methods s h o u l d have an a v e r a g e

po tency t i t e r

v a l u e a s fo l lo w s:

a . t l e a s t a 1 r e a c t i o n w i t h a 1:8 d i l u t i o n o f

r e a g e n t

Anti-K

Anti-k

Ant i - Jka

An t i -Fya

Anti-Cw

b . t l e a s t a 1 r e a c t i o n w i t h a 1 : 4 d i l u t i o n o f

r e a g e n t

c . t l e a s t a 2 r e a c t i o n w i t h u n d i l u t e d r e a g e n t :

Anti-U

Anti-Kpa

Anti-Kpb

Ant i - J sa

Ant i - J sb

Anti-Fyb

Anti-N

Anti-Lea

Anti-Leb

Anti-Dia

Ant i - M q

Anti-Jkb

Anti-Xga

Anti-Cob

Anti-Wr"

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DR FT

2. Productsofmonoclonaloriginwhicharerecommended

for tube test methods should have an average

potency titervalue of at least 1+ with a

1:8

dilutionofreagent.

Manufacturersthatwishtoestablishpotencytiter

valuesotherthanthesearetoobtainapprovalfrom

theDirector,CenterforBiologicsEvaluationand

Researchatthetimeoflicenseapplication.

3. Products recommended for use in automated or

microplate systems without user dilution (as

supplied)shouldbesufficientlypotentthat

two-

fold dilutionprepared with anapproveddiluent

willproducethesamequalitativetestresultas

the undiluted productwhentested in accordance


111.SPECIFICITYTESTING

A. REAGENTDILUTIONS






1. Anycellsofanyagemaybeusedinthe"Testto

Confirm Reactivity with Antigen Positive Cellsvv

(III.C.l). Inthe"TesttoConfirm Absence of

Contaqinating Antibodies" (III.C.2) licensed


theirexpirationdate. All otherredbloodcell

samplesshouldbeusedwithin

7

daysofcollection

fromthedonor.

Manufacturersthatwishtousecellsmorethan

7


approvalfromtheDirector,CenterforBiol.ogics



2. Redbloodcellsmeetingthecriteriaofparagraph1


the preparationof cell suspensionsforreagent




thethawedredbloodcellsonthedayofuse, In

the case of cells expressing low frequency



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DRAFT

Themethod of freezing,storing,andthawingred




~iologicsEvaluation and Researchbefore use in


3.

Eachbatchof redbloodcellsforuse

in

c!ontrol





4. Licensed reagentredblood cellsmay be used as

provided.







1.


CELLS

a. At least4different

donorswhoseredblood

cellsexhibitweakor

heterozygousexpression




should be confirmed separately by using 4

differentcellspossessing only one of the

antigensforeachdifferentspecificity.



negativecontrol.

2. TESTTOCONFIRMABSENCEOFCONTAMINATINGANTIBODIES

Testthereagentforthepresence of antibodies

correspondingtothefollowingantigensby oneof


A,B H,Lea,Leb,

I K

k,Kp",Kpb,Jsb,PI,D,C,

E, c,e,Cv,M,N,S,s,U,Lu",Lub,Jk",JklD,Fy",

Fyb,Xg",Do",Dob,Yt",Ytb,Lan,Con,cob,MY,Wr",

andSd".

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DRAFT

Ifthesourcematerial isa monoclonal antibody



A

B,H Lea,Leb,I,K k,K>, JsbI,,D,C E,

c,

e,M I

N S

s,

U

Lub,Jk",Jkb,Fy",andFyb


includedon thislistmaybe requested from the



submissi/onofthefirstprotocol.

a. Performa direct test for the presence of


cellsfromatleast

4

differentdonorswhose


reagentantibody.

When red blood cells lacking the antigen




knownphenotype.

Theadsorbedserummaythenbetestedagainst

red blood cellswhich exhibitany antigens



subsequenttestingshouldbeapprovedby the


Research.

.When direct tests are impractical, the



antibodiesmay bepresumptivelyexclutded by




antibodies.

Red blood cell samplesfrom fourdifferent


the absenceof contaminating antibodies to


99 inthegeneralpopulationof theUnited

States.

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DRAFT

3.

TESTTOCONFIRMABSENCEOFANTI-AANDANTI-B

a. GroupA,and Bredblood cellslackingthe


shouldbetested. GroupA,B redbloodcells

maybesubstitutedforA,and/orBredblood


Adsorbed serummay be used as inIII.C.2.b

ao.ve

4.

ADDITIONALPHENOTYPESRECOMMENDEDFORTESTING


Anti-A, A,,A,,A,B, A,B,B,and0

Anti-Leb

6

differentA,and/orA,BLe(b-I)

Anti-P, Atleast2P,weak(asdeterminedby

titrationstudies)

GroupAandABcellswhichdoreactwithanti-A,

anddonotreactwithanti-'H.

THETESTS

1. Toconfirmreactivitywithantigenpositivecells,




Minimum parameters (dropsof reagent,incubation

time,centrifugation,etc.)shouldbefollowed.

2. To confirm absence of contaminating antibodies,



method(s)describedinthemanufacturer's package

insert. Maximum parameters (drops of reingent,

incubationtime,centrifugation,etc. shou~ldbe

followed.

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DRAFT





Red blood cells which exhibit the--=antigen


atleasta2+reaction.

Ifanyofthefoursamples

testedyieldslessthana 2+reaction,redblood




cellsamplesyieldslessthana2+ reactionwith

thetestreagent.

Whentestingunusualphenotypes,

othercriteriafor

reactivitymayapply. Forexample,Fyxredblood

cellsmaynotyielda2+reactionwithAnti-Fybbut

shouldyieldaclearlypositivemacroscopicresult.

3. Thenegativecontrolcell(s)instepIII.C.1should

yield a negative reaction by each test nnethod



correspondingto the reagentantibody and tests

with adsorbed reagent should be negative, thus



III.C.2

5.

Themanufacturer should list on the lotrelease

protocol and in the "Specific Performance

Characteristics*'section of the package insert

thoseredbloodcellantigenslistedinIII.C.2for


If desired,theredbloodcell phenotypeof the

antibodyhonor(s)mayalsobelistedaspresunnptive


present.

6.

TestswithgroupA andBredbloodcellsshouldbe

negative,thusconfirmingtheabsenceofanti--And

anti-B.

7. Confirmation by the manufacturer of nonspc'cific

reactionsafteralotofBloodGroupingReagenthas


manufacturertotheDirector,CenterforBiologics


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DRAFT


A. REAGENTDILUTIONS

1.

Preparea1

in2dilutionofthereagentundertest

bymixingequalpartsofthereagentandABserum,

groupcompatibleserum,oradiluentapprovedby

theDirector,Centerfor~iologics

v lu tion

and

Research.


1.

Redbloodcellsshouldbepreparedaccordingtothe



1. Redbloodcellsfromatleasttwodifferentdonors

exhibitingweakand/orheterozygousexpressionof

theantigencorrespondingtothereagentantibody

shouldbeused.


1. Thetest istobeperformedwith both undiluted

reagentandthedilutedreagentprepared in step

IV,A by the method recommended in the



1. Testresultsareobservedandrecordedatonehalf


andaftheendofthefullrecommendedobservation

time

F . AVIDITYTESTINGRESULTS

1.


theundilutedanddilutedreagentatonehalf of

themanufacturer's recommendedobservationtime.



endofthemanufacturer's recommendedobservation

timeandshouldbereportedasgreaterthanorless

than1 mm.

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UCM062695.pdf