I DOCKET SO.

8 4 s - 0 1 8 1 i

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 20892
301-227-6487.

Submit vritten comments on this draft to:

Dockets Hanagement Branch ( H F A - 3 0 5 )

Food and Drug Administration
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 A £ fairs Branch ( HFB-1 4 2 )

Food and Drug Administration
5600 Fishers Lane
Rockville. YD 20857
301-295-8228
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 109. Yletro Park Sorth 3
7 5 6 4 Standish Place
Rochille. YD 20855.

Comments and requests should be identified with the docket number found in
brackets in the heading of this document.
 DRAFT

PROPOSED R E V I S I O N

RECOMMENDED METHODS FOR

BLOOD GROUPING REAGENTS EVALUATION

MARCH 1 9 9 2

TABLE O F CONTENTS

SUMMARY

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

PROPOSED RECOMMENDED METHODS

ABO BLOOD GROUPING REAGENTS

REFERENCE PREPARATIONS .............................1

POTENCY T E S T I N G ....................................2

SPECIFICITY TESTING ................................ 5

AVIDITY T E S T .......................................
9
T E S T FOR SPONTANEOUS AGGLUTINATION.. . . - . - . - - - - .11
..-
S L I D E AND M O D I F I E D TUBE RH BLOOD GROUPING REAGENTS

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

...............................1 3

POTENCY T E S T I N G . . . .
SPECIFICITY TESTING.................. .............1 6

AVIDITY T E S T . . ....................................2 2

T E S T FOR PROZONE ..................................2 3

'I'EST FOR PROZONE - .......- 2 5

METHOD 2...............

LOW PROTEIN RH BLOOD GROUPING REAGENTS

REFERENCE PREPARATIONS ............................2 7

.....................2 8

POTENCY T E S T I N G . . . . . . . . . . . . . .
SPECIFICITY TESTING ............................... 3 2

.............................38

AVIDITY T E S T . . . . , . . . .
T E S T FOR SPONTANEOUS AGGLUTINATION ................40

T E S T FOR PROZONE ..................................40

T E S T FOR PROZONE - METHOD 2..............-....-...42

RARE BLOOD GROUPING REAGENTS

REFERENCE PREPARATIONS ............................

44

POTENCY T E S T I N G ...................................

44
...............................4 8

S P E C I F I C I T Y TESTING
AVIDITY TEST ......................................

53

 - --

DRAFT
ABO BLOOD GROUPING REAGENTS

These recommended methods are provided to help assist manufacturers

in pursuing new product license applications and amendments to

existing product license applications. The methods ,described

herein do not bind the agency, and manufacturers may consider use

of other methods. In cases where manufacturers wish to use methods

other than those described herein, FDA recommends that the matter

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

values, specificity results, and other matters referred to in this

document are intended to assist manufacturers in preparing

submissions to FDA. The information is based on current knowledge

and is not meant to be all inclusive and should not be viewed as

ensuring approval or the only means of achieving FDA approval.

Following the methods provided in this document will assist in the

approval process, but does not guarantee approval. FDA will review

applications on an individual basis and approvals will be granted

when supported by scientific evidence.

I. REFERENCE PREPARATIONS

A. The Reference Blood Grouping Reagents listed below can be

obtained from:

Center for Biologics Evaluation and Research

Food and Drug Administration

8800 Rockville Pike

Bethesda, MD. 20892

USA

NOTE: FDA Reference Blood Grouping Reagents are not

routinely available to anyone except U.S. licensed

manufacturers and amounts issued will be

proportional to lots released in the previous year.

B. Reference sera are to be used according to the

accompanying package insert a for determining the

potency of Blood ~roupingReagents as part of their final

lot release testing.

In-house reference materials should be developed for all

stability testing, in process testing or product

development purposes.

 DRAFT

11. POTENCY TESTING

A. REAGENT DILUTIONS

1. Beginning with the undildted reagent, prepare

separate master two-fold serial dilutions (1 in 2,
1 in 4, etc. ) of the test reagent using isotonic
saline containing 1-2% bovine albumin -or=another
diluent approved by the Director, Center for
Biologics Evaluation and Research. Test tubes
should be of a size that facilitates adequate
mixing of the contents (12 X 75 mm or larger).
If the endpoint is expected to exceed 1024,

accuracy will be improved if direct intermediate

dilutions are done to keep the number of serial

transfers to less than 10. (e.g., If the expected

endpoint is 4096, prepare an initial 1:10 dilution

with the same diluent as used above.)

NOTE : All titrations should be carried to a

negative endpoint. (See E.4)
2. Prepare master dilutions of the Reference Blood

Grouping Reagent(s) as in paragraph 1 of this

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

dilutions of each Reference Blood Grouping Reagent

separately.

3. A separate, clean pipet or pipet tip should be used

for each dilution (including any intermediate
dilutions) to avoid carryover of higher reagent
concentrations.
4. The last tube should contain diluent only and serve

as a diluent control.

B. RED BLOOD CELL PREPARATIONS

Fresh or frozen red blood cells may be used for preparing

cell suspensions for the potency testing of all Blood

Grouping Reagents under the following conditions:

1. Red blood cells of any age may be used, provided

the titer values of the reference reagents are

within an acceptable range.

2. Red blood cells may be frozen and thawed for use in

the preparation of cell suspensions for .reagent

evaluation. To ensure that the correct cell has

been thawed, appropriate controls should be used to

demonstrate the desired reactivity and identity of

the thawed red blood cells on the day of use.

 DRAFT
The method of freezing, storing, and thawing red

blood cells, including a description of the

cryoprotective medium should be described in detail

and should bk approved by the Director, Center for

Biologics valuation and Research before use in

control testing of licensed reagents.

3. Red blood cells should be washed at leas~=twicein

isotonic sa$ine or until a clear supernate is

obtained and then resuspended to a 2% suspension in

isotonic sallinecontaining 1-2% bovine albumin or

another dilqent approved by the Director, Center

for Biologicb Evaluation and Research.

C. MINIMUM TEST CELL6 FOR POTENCY

REAGENT RED BLOOD CELLS

Anti-A A, and 3 DIFFERENT A,B *

Anti-B B and A,B

Anti-A,B A,, A, **, and B

Anti-A and B A,, A, **, and B

* AB cells which do not react with anti-A, and do

react with ahti-H.
** A cells dhich do not react with anti-A, and do
react with ahti-H.
D. THE TEST (BY TUBE METHOD)

1. Place 0.1 milliliter of each reagent dilution in a

separate, cl$an test tube (approximately 10 X 7 5 mm
or 12 X 7 5 mrh).
2. Place 0.1 ailliliter of each Reference Blood
Grouping Reagent dilution in a separate, clean test
tube (approx+mately 10 X 7 5 nun or 12 X 7 5 mm).
3. Add 0.1 milliliter of the appropriate 2% cell

suspension tb each test tube.

4. Mix the contqnts of each tube thoroughly. Incubate

for 5 minute$ at room temperature (RT; 20-30 C) and

centrifuge for 1 minute at approximately 1000 rpm

(100-125 rcf) or 15 seconds at approximately 3400

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

appropriate for the centrifuge being used.

 DRAFT

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 clumps.

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 granules, 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.
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 considered as

contributing to the dilution of the reagent.

4. Test results should include at least one tube with

no agglutination after the endpoint. The diluent

control tube should be negative.

F. POTENCY TITER VALUES

1. ABO Reagents should have an average potency titer

value at least equal to that of the reference

reagent.

2. Products recommended for use in automated or

microplate systems without user dilution (as

supplied) should be sufficiently potent that a two-

fold dilution prepared with an approved diluent

will produce the same qualitative test result as

the undiluted product when tested in accordance

with the manufacturer's package insert.

 DRAFT

111, SPECIFICITY TESTING

A. REAGENT DILUTIONS

1. No dilution of the reagent under test is permitted.

B. RED BLOOD CELL PREPARATIONS

Fresh or frozen red blood cells may be used f ~ r ' ~ r e ~ a r i n g

cell suspensions for the specificity testing of all Blood

Grouping Reagents under the following conditions:

1. Any cells of any age may be used in the "Test to

Confirm Reactivity with Antigen Positive Cells1@

(IIIC.1). In the "Test to Confirm Absence of

contaminating Antibodiesw (III.C.2) licensed

reagent red blood cells may be used any time before

their expiration date. All other red blood cell

samples should be used within 7 days of collection

from the donor.

Manufacturers that wish to use cells more than 7

days after collection from the donor are to obtain

approval from the Director, Center for Biologics

Evaluation and Research, and are to provide

sufficient data to support the request.

2. Red blood cells meeting the criteria of paragraph 1

of this section may be frozen and thawed for use in

the preparation of cell suspensions for reagent

evaluation. To ensure that the correct cell has

been thawed, appropriate controls should be used to

demonstrate the desired reactivity and identity of

the thawed red blood cells on the day of use. In

the ,case of cells expressing low frequency

antigens, testing for several common antigens may

serve to adequately identify the cell.

The method of freezing, storing, and thawing red

blood cells, including a description of the

cryoprotective medium should be described in detail

and should be approved by the Director, Center for

Biologics Evaluation and Research before use in

control testing of licensed reagents.

Licensed reagent red blood cells may be used as

provided.

All other red blood cell samples should be washed

at least twice in isotonic saline or until a clear

supernate is obtained and then resuspended with an

approved diluent to the cell concentration listed

in the manufacturer's package insert.

 DRAFT

- - . - -
C. MINIMUM TESTING FOR SPECIFICITY

1. TEST TO CONFIRM REACTIVITY WITH ANTIGEN POSITIVE

CELLS

a. At least 4 different donors whose red blood

cells exhibit expression of the antigen should
be tested. . -
b. When testing Anti-A,B and Anti-A and B

reagents, the reactivity with both group A and

group B red blood cells should be confirmed

separately, i,e, at least four group A donors

should be used to confirm the reactivity of

the Anti-A component and at least four group B

donors should be used to confirm the

reactivity of the ~ n t i - Bcomponent.

c. Minimum test red blood cells recommended:

REAGENT RED BLOOD CELLS

Anti-A A, (1) and A,B (3) *

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

Anti-A,B A, (21, A, ** (21, B (411

at least 3 different A, ***
Anti-A and B A, (2), A, ** (2), B (4),
at least 3 different A, ***
* AB cells which do not react with anti-A, and
do react with anti-H.
* * A cells which do not react with anti-A, and
do react with anti-H.
*** A, cells are recommended; labeling should
indicate detection of group A variants.
Include examples of I1strong & cellsIf and
I1moderate & cells1'. "Weak & cellsIf are
optional.
d. Include at least one red blood cell which does

not exhibit expression of the antigen as a

negative control.

 --
2. TEST TO CONFIRM-ABSENCE OF CONTAMINATING ANTIBODIES
Test the reagent for the presence of antibodies

corresponding to the following antigens by one of

the methods listed below.

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

E, c, e, Cw, M, N, S , s , U, Lu", Lub, Jksl. Jkb, Fyal

.
F Y ~ ,Xg", DO", Dob, Yt", YtbI Lan, Co", Cob;'Mg, Wrar

Sd", and Vw.

If the source material is a monoclonal antibody

from a previously characterized and licensed clone,

this list may be shortened as follows:

A, B, H, Lea, Leb, I, K, k, Kpb, Jsb,PI, D, C, E, c,

e, M, N, S, s, U, Lub, Jksl Jkb, FY", and Fyb.

Approval for the use of fewer antigens than

included on this list may be requested from the

Director, Center for ~iologics Evaluation and

Research by a manufacturer at the time of

submission of the first protocol.

a, Perform a direct test for the presence of

contaminating antibodies by using red blood

cells from at least 4 different donors whose

cells lack the antigen corresponding to the

reagent antibody.

b. When red blood cells lacking the antigen

corresponding to the reagent antibody under

test are not available, the reagent antibody

may be adsorbed to exhaustion with cells of a

known phenotype.

The adsorbed serum may then be tested against

red blood cells which exhibit any antigens

which were not present on the cells used for

adsorption. The methods for adsorption and

subsequent testing should be approved by the

Director, Center for Biologics Evaluation and

Research,

c. When direct tests are impractical, the

Director, Center for Biologics Evaluation and

Research may approve procedures whereby

antibodies may be presumptively excluded by

testing an appropriate number of non-reactive

red blood cell samples to provide statistical

assurance of the absence of contaminating

antibodies.

 DRAFT
d. Red blood cell samples from four different

donors may be used to confirm presumptively

the absence of contaminating antibodies to

antigens having an incidence of greater than

99% in the general population of the United

States or the country in which it is sold.

D. THE TESTS . . .-
1. To confirm reactivity with antigen positive cells,

each lot of Blood Grouping Reagent should be tested

and results interpreted by all test methods

described in the manufacturer's package insert.

Minimum parameters (drops of reagent, incubation

time, centrifugation, etc.) should be followed.

2. To confirm absence of contaminating antibodies,

each lot of Blood Grouping Reagent should be tested

and results interpreted by the most sensitive test

method(s) described in the manufacturer's package

insert. Maximum parameters (drops of reagent,

incubation time, centrifugation, etc.) should be

followed.

E. SPECIFICITY RESULTS

1. No hemolysis or rouleaux formation should be

detected by any of the methods in the

manufacturer's package insert.

Red blood cells which exhibit the antigen

corresponding to the reagent antibody should yield
at least a 2+ reaction. If any of the four samples
tested yields less than a 2+ reaction, red blood
cells from four additional donors who exhibit the
antigen should be tested. The test is considered
satisfactory if no more than one of eight red blood
cell samples yields less than a 2+ reaction with
the test reagent.
When testing unusual phenotypes, other criteria for

reactivity may apply. For example, a larger

percentage of A, red blood cells may not yield a 2+

reaction with Anti-A,B and Anti-A and B but should

yield a clearly positive macroscopic result.

3. The negative control cell(s) in step II1.C. 1 should

yield a negative reaction by each test method

described in the manufacturer's package insert.

4. Tests with red blood cells which lack the antigen

corresponding to the reagent antibody and tests

with adsorbed reagent should be negative, thus

confirming the absence of significant contaminating

 DRAFT
- Y -.d :- - : =---=. -4

4. Tests with red blood cells which lack the antigen

corresponding to the reagent antibody and tests

with adsorbed reagent should be negative, thus

confirming the absence of significant contaminating

antibodies directed at the antigens listed in

III.C.2

5. The manufacturer should list on the l ~ t - ~ r e l e a s e

protocol and in the n~pecific Performance

Characteristicsn section of the package insert

those red blood cell antigens listed in III.C.2 for

which no specificity tests have been performed.

If desired, the red blood cell phenotype of the

antibody donor(s) may also be listed as presumptive

evidence that antibodies to those factors are not

present.

6 Confirmation by the manufacturer of n0nspeci:fi.c

reactions after a lot of Blood Grouping Reagent has

been released should be reported promptly by the

manufacturer to the Director, Center for Biologics

Evaluation and Research.

IV. AVIDITY TEST FOR SLIDE REAGENTS

A. REAGENT DILUTIONS

1. Prepare a 1 in 2 dilution of the reagent under test

by mixing equal parts of the reagent and AB serum

which is free of antibodies or a diluent approved

by the Director, Center for Biologics Evaluation

and Research.

B. RED BLOOD CELL PREPARATIONS

1. Red blood cells should be prepared according.to the

manufacturer's package insert.

C. MINImBl TEST CELLS FOR AVIDITY

REAGENT RED BLOOD CELLS

Anti-A A,, and A,B *

Anti-B B and A,B

Anti-A,B A,, A, **, B, and & *** .

.. .
,.
.
-
Anti-A and B A,, A, **, B, and & ***
* AB ce1l.s which do not react with anti-A, and do
react with anti-H.
** A ce1l.s which do not react with anti-A, and do
react with anti-H.
*** A, red blood cells are recommended only if the
reagent is recommended for detection of weak
subgroups of A by slide technique.
D. THE TEST (BY SLIDE METHOD)

1. The test is to be performed with both undiluted

reagent and the diluted reagent prepared in step

1V.A by the method recommended in the

manufacturer's package insert.

E. INTERPRETATION OF THE TEST

1- Test results are observed and recorded at one half

of the manufacturer's recommended observation time

and at the end of the full recommended observation

time.

F . AVIDITY TESTING RESULTS

1. Signs of agglutination should be observed with both

the undiluted and diluted reagent at the end of the

first half of the observation time.

2- Clear maciroscopic agglutination should be observed

with both the undiluted and diluted reagent at the

end of the observation time and should be reported

as greater than or less than 1 mm in diameter.

 DRAFT

V. TEST FOR SPONTANEOUS AGGLUTINATION

A. REAGENT DILUTIONS

1. No dilution of the reagent under test is permitted.

B. RED BLOOD CELL PREPARATION

1. Obtain c positive group 0 red blood cells"-@ram one

donor.

2. Coat the red blood cells heavily with an IgG anti-c

such that a 3-4+ direct antiglobulin test (DAT) is

achieved and positive reactions are obtained with a

high protein Rh control reagent, but negative

reactions are obtained with a saline control.

The exact procedure for coating the red blood cells

will depend on the specific antibody chosen for

coating and its strength.

C. THE TEST

1. Test the coated cell sample according to the

manufactu:rer8spackage insert.

D. INTERPRETATION

1. Blood Grouping Reagents for use by a low protein

tube test method should not spontaneously

agglutinate immunoglobulin coated red blood cells.

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 blood

group results should be recommended or supplied.

3. If a control test or reagent is recommended by the

manufacturer, cells for use in control testing in

sections 11, 11, and IV should give a negative

direct antiglobulin test (DAT).

- -7 ; SLIDE*-AND MODIFIED TUBE RH BLQOD ,GRQ?PJK REAGENTS - DRAFT

-
d

GENE,FIAL INFORMATION

These recommended methods are provided to help assist manufacturers

in pursuing new product license applications and amendments to

existing product license applications. The methods described

herein do not bind the agency, and manufacturers may cons.ider use

of other methods. In cases where manufacturers wish to use'methods

other than those described herein, FDA recommends that the matter

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

values, specificity results, and other matters referred to in this

document are intended to assist manufacturers in preparing

submissions to FDA. The information is based on current knowledge

and is not meant to be all inclusive and should not be viewed as

ensuring approval or the only means of achieving FDA approval.

Following the methods provided in this document will assist in the

approval process, but does not guarantee approval. FDA will review

applications on an individual basis and approvals will be granted

when supported by scientific evidence.

I. REFERENCE PREPARATIONS

A. The Reference Blood Grouping Reagents listed below can be

obtained from:
Center for Biologics Evaluation and Research

Food and Drug Administration

8800 Rockville Pike

Bethesda, MD. 20892

USA

Anti-D for evaluation of IgG products

Anti-c for rapid tube test

Anti-E for rapid tube test

Anti-c for rapid tube test

Anti-e for rapid tube test

NOTE : FDA Reference Blood Grouping Reagents are not

routinely available to anyone except U.S.

licensed manufacturers and amounts issued will

be proportional to lots released in the

previous year.

B. All reference sera are to be used according to the

accompanying package insert only for determining the

potency of Blood Grouping Reagents as part of their.final

lot release testing.

 DRAFT

In-house reference materials should be developed for all

stability testing, in process testing or product

development purposes.

11. POTENCY TESTING

A. REAGENT DILUTIONS

-
. .
1. Beginning with the undiluted reagent, prepare
separate master two-fold dilutions (1 in 2, 1 in 4,
etc. ) of the test reagent using 20-22% bovine
albumin or another diluent approved by the
Director, Center for ~iologics Evaluation and
Research. Test tubes should be of a size that
facilitates adequate mixing of the contents (12 X
75 mm or larger).

f the endpoint is expected to exceed 1024,

.ccuracy will be improved if direct intermediate

.ilutions are done to keep the number of serial

transfers to less than 10. (e.g., If the expected

endpoint is 4096, prepare an initial 1:10 dilution

with the same diluent as used above.)

NOTE : All titrations should be carried to a

negative endpoint. (See E.4)
2. Prepare master dilutions of the Reference Blood

Grouping Reagent(s) as in paragraph 1 of this

section. For test reagents containing multiple

antibodies (ex. ~nti-CDE)dilutions of each of the

corresponding Reference Blood Grouping Reagents

should be made separately.

3. A separate, clean pipet or pipet tip should be used

for each dilution (including any intermediate
dilutions) to avoid carryover of higher reagent
concentrations.
4. The last tube should contain diluent only and serve

as a diluent control.

B. RED BLOOD CELL PREPARATIONS

Fresh or frozen red blood cells may be used for preparing

cell suspensions for the potency testing of all Blood

Grouping Reagents under the following conditions:

1. Red blood cells of any age may be used, provided

the titer values of the Reference Blood Grouping

Reagent(s) are within an acceptable range.

 DRAFT
2. Red blood cells may be frozen and thawed for use in

the preparation of cell suspensions for reagent

evaluation. To ensure that the correct cell has

been thawed, appropriate controls should be used to

demonstrate the desired reaztivity and identity of

the thawed red blood cells on the day of use.

The method of freezing, storing, and thauing red

blood cells, including a description of the

cryoprotective medium should be described in detail

and should be approved by the Director, Center for

Biologics valuation and Research before use in

control testing of licensed reagents.

3. Each batch of red blood cells for use in control

testing of reagents requiring indirect antiglobulin

technique should be tested for absence of

complement or immunoglobulin molecules (DAT

negative) on the day of use.

4. Red blood cells should be washed at least twice in

isotonic saline or until a clear supernate is

obtained and then r2suspended to a 2% suspension in

isotonic saline containing 1-2% bovine albumin or

another diluent approved by the Director, Center

for Biologics Evaluation and Research.

C. MINIMUM TEST CELLS FOR POTENCY

REAGENT RED BLOOD CELLS

Anti-D Dce

( Ror 1

dCce
(r'r) .

Anti-E dcEe

(rl1r)

Anti-c DCcEe

( R,R,

Anti-e dcEe

(rl1r)

Anti-CD dCce and Dce

(r'r and Ror)

Anti-DE Dce and dcEe

(Rorand r1Ir)

Anti-CDE dCce and Dce and dcEe

(rtr and R,,r and rVtr)

D. THE TEST (BY TUBE METHOD)

1. Place 0.1 milliliter of each reagent dilution in a

separate, clean test tube (approximately 10 X 7 5 mm

or 12 X 7 5 mm).

2. Place 0.1 milliliter of each Reference Blood

Grouping Reagent dilution in a separate, ciean test

tube (approximately 10 X 75 mm or 12 X 7 5 mm).

3. Add 0.1 milliliter of the appropriate 2% cell

suspension to each test tube.

4. Mix the contents of each tube thoroughly and

incubate the test tubes for 15 minutes at 37 C.

5. -Centrifuge for 2 minutes at approximately 1000 rpm

(100-125 rcf) or 45 seconds at approximately 3400

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

appropriate for the centrifuge being used.

E. INTERPRETATION OF THE TEST

1. The cell buttons of each test tube shou1.dbe 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 clumps.

2+ Cell button dislodges into many sinall clumps

of equal size.

1+ Cell button dislodges into finely granular,

but definite, small clumps.

D Cell button dislodges into fine granules, 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 int.0

no visible clumps.

3. The potency titer value is the reciprocal of the

greatest reagent dilution for which the reaction is

graded at I+.

 DRAFT

- --a%& '- u7 4Th+-dilution -caused4by Wie addition of -%fie rep'; ?F

blood cells should not be considered 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. Slide and Modified Tube Rh Blood Grouping Reagents

should have an average potency titer value at least

equal to that of the reference reagent.

2. Products recommended for use in automated or

microplate systems without user dilution (as

supplied) should be sufficiently potent that a two-

fold dilution prepared with an approved diluent

.will produce the same qualitative test result as

the undiluted product when tested in accordance

with the manufacturer's package insert.

111. SPECIFICITY TESTING

A. REAGENT DILUTIONS

1. No dilution of the reagent under test is permitted.

B. RED BLOOD CELL PREPARATIONS

Fresh or frozen red blood cells may be used for preparing

cell suspensions for the specificity testing of all Blood

Grouping Reagents under the following conditions:

Any cells of any age may be used in the "Test to

Confirm Reactivity with Antigen Positive Cellsw

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

Contaminating Antibodies" (III.C.2) Licensed

reagent red blood cells may be used any time before

their expiration date. All other red blood cell

samples should be used within 7 days of collection

from the donor.

Manufacturers that wish to use cells more than 7

days after collection from the donor are to obtain

approval from the Director, Center for Biologics

Evaluation and Research, and are to provide

sufficient data to support the request.

2. Red blood cells meeting the criteria of paragraph 1

of this section may be frozen and thawed for use in

the preparation of cell suspensions for reagent

evaluation. To ensure that the correct cell has

been thawed, appropriate controls should be used to

 - 1- -
'
P
- *.+;
T f B e --i
, , g r e -
DRAFT
rxsmE
demonstrate the desired reactivity and identity of

- -cr2

the thawed red blood cells on the day of use. In

the case of cells expressing low frequency

antigens, testing for several common antigens may

serve to adequately identify the cell.

The method of freezing, storing, and thawing red

blood cells, including a description of the _
cryoprotective medium should be described'i-n detail
and should be approved by the Director, Center for
~iologics Evaluation and Research before use in
control testing of licensed reagents.
3. Each batch of red blood cells for use in control

testing of reagents requiring indirect antiglobulin

technique should be tested for absence of

complement or immunoglobulin molecules (DAT

negative) on the day of use.

4. Licensed reagent red blood cells may be used as

provided.

All other red blood cell samples should be washed

at least twice in isotonic saline or until a clear

supernatant is obtained and then resuspended with

an approved diluent to the cell concentration

listed in the manufacturer's package insert.

C. MINIMUM TESTING FOR SPECIFICITY

1. TEST TO CONFIRM REACTIVITY WITH ANTIGEN POSITIVE

CELLS

a. At least 4 different donors whose red blood

cells exhibit weak or heterozygous expression

of the antigen should be tested.

b. When testing reagents containing multiple

antibodies, the reactivity of each specificity

should be confirmed separately by using 4

different red blood cells possessing only one

of the antigens for each different

specificity.

ex. For Anti-CDE reagents, at least four

donors should be used to confirm the

reactivity of the Anti-C component, at least

four donors should be used to confirm the

reactivity of the Anti-D component, and at

least four donors should be used to confirm

the reactivity of the Anti-E component.

-- -------- --A
- -
DRAFT ''---

c. Include at least one red blood cell which does

not exhibit the expression of the antigen as a

negative control.

TEST TO CONFIRM ABSENCE OF CONTAMINATING ANTIBODIES

Test the reagent for the presence of antibodies

corresponding to the following antigens by one of

the methods listed below.

.. .-
A, B, H, Lea, Leb, I, K, k , Kpa, Kpb, Jsbt P,, D, C,

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

FybI XgaI Doa, Dob, Ytal YtbI Lan, Coal Cob, Mq, Wra,

and Sda.

If the source material is a monoclonal antibody

from a previously characterized and licensed clone,

this list may be shortened as follows:

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

e, M, N, S, s, U, Lub, Jkal Jkb, Fya, and Fyb.

Approval for the use of fewer antigens than

included on this list may be requested from the

Director, Center for Biologics Evaluation and

Research by a manufacturer at the time of

submission of the first protocol.

a. Perform a direct test for the presence of

contaminating antibodies by using red blood

cells from at least 4 different donors whose

cells lack the antigen corresponding to the

reagent antibody.

b. When red blood cells lacking the antigen

corresponding to the reagent antibody under

.test are not available, the reagent antibody

may be adsorbed to exhaustion with cells of a

known phenotype.

The adsorbed serum nay then be tested against

red blood ce'lls which exhibit any antigens

which were not present on the cells used for

adsorption. The methods for adsorption and

subsequent testing should be approved by the

Director, Center for Biologics Evaluation and

Research.

c. When direct tests are impractical, the

Director, Center for Biologics valuation and

Research may approve procedures whereby

antibodies may be presumptively excluded by

testing an appropriate number of non-reactive

red blood cell samples to provide statistical

 DRAFT

assurance of the absence of contaminating

antibodies.

d. Red blood cell samples from four different

donors may be used to confirm presumptively
the absence of contaminating antibodies to
antigens having an incidence of greater than
99% in the general population of the United
, *
States.
3. TEST TO CONFIRM ABSENCE OF ANTI-A AND ANTI-B

a. Group A, and B red blood cells lacking the

antigen corresponding to the reagent antibody

should be tested. Group A,B red blood cells

may be substituted for A, and/or B red blood

cells if either are unavailable.

Adsorbed serum may be used as in III.C.2.b

above.

4. PHENOTYPES RECOMMENDED FOR TESTING

As a minimum, red blood cells exhibiting the

following phenotypes should be used in the

specificity testing outlined in steps 1, 2, and 3

above.

MINIMUM RED BLOOD CELLS

DCce, Dce, dCce, and dcEe

(R,r, Ror, r t r , and rMr)

A, dce, B dce, and 0 dce (rr)

Vw positive

3 different dce (rr) Bg(a+) *

6 Du samples representing different
Rh phenotypes and reactive by the
Indirect Antiglobulin Test only *
Anti-D Category IV, V, and VI cells

(monoclonal)

Dce, dCce, and dcEe or dcE

( R r , r t r , and rI1r or r"r'l)

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

Cw positive (e.g. RlUr)

A, dce, B dce, and 0 dce (rr)

Dce, dCce or dCe, and dcEe

( R r , r t r or r t r t, and rV1r)

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

A, dce, B dce, and 0 dce (rr)

 DRAFT

dCce and DCEe or DCE or dCE

(r'r and R,R, or R,R, or rYrY)

A, DCe, B DCe, and 0 DCe (R,R,)

DCcEe f neg (R,R2)

-
Anti-e dcEe and DCcE or DCE or dCE

(rI1rand R,R, or R,R, or rYrY)

A, DcE, B DcE, and 0 DcE (R,RR,.

DCcEe f neg (R,R,)

Anti-CD Dce, dCce, and dcE or dcEe

(R,r, r' r, and r1Irl1or rItr)
A, dce, B dce, and 0 dce (rr)
rCr or rUCr ***
Anti-DE Dce, dCce or dCe, and d c ~ e

(Kr, r'r or r'rf,and rI1r)

A, dce, B dce, and 0 dce (rr)

Dce, dCce, and dcEe

(Kr, r'r, and rtgr)

A, dce, B dce, and 0 dce (rr)

rCr ***

* For Anti-D reagents recommended for D" testing

** rf"rcells may be used in addition to but not

as a substitute for C+ Ce- cells
*** Recommended if labeling indicates detection of
G antigen

D. THE TESTS .
1. To confirm reactivity with antigen positive cells,

each lot of Blood Grouping Reagent should be tested

and results interpreted by all test methods

described in the manufacturer's package insert.

Minimum parameters (drops of reagent, incubation

time, centrifugation, etc.) should be followed.

2. To confirm absence of contaminating antibodies,

each lot of Blood ~roupingReagent should be tested

and results interpreted by the most sensitive test

method(s) described in the manufacturer's package

insert. Maximum parameters (drops of reagent,

incubation time, centr.ifugation, etc.) should be

followed.

 DRAFT

E. SPECIFICITY RESULTS

1. No hemolysis or rouleaux formation should be

detected by any of the methods in the

manufacturer's package insert.

2. Red blood cells which exhibit the antigen

corresponding to the reagent antibody should yield
at least a 2+ reaction. If any of the four'samples
tested yields less than a 2+ reaction, red blood
cells from four additional donors who exhibit the
antigen should be tested. The test is considered
satisfactory if no more than one of eight red blood
cell samples yields less than a 2+ reaction with
the test reagent.
When testing unusual phenotypes, other criteria for

reactivity may apply. For example, a larger

percentage of C+ Ce- red blood cells may not yield

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

clearly positive macroscopic result.

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
yield a negative reaction by each test method
described in the manufacturer's package insert.
4. Tests with red blood cells which lack the antigen
corresponding to the reagent antibody and tests
with adsorbed reagent should be negative, thus
confirming the absence of significant contaminating
antibodies directed at the antigens listed in

5. The manufacturer should list on the lot release

protocol and in the "specific Performance

Characteristics" section of the package insert

those red blood cell antigens listed in I11 .C.2 for

which no specificity tests have been performed.

If desired, the red blood cell phenotype of the

antibody donor(s) may also be listed as presumptive

evidence that antibodies to those factors are not

present.

6. Tests with group A, and B red blood cells should be

negative, thus confirming the absence of anti-A and
anti-B.
7. Confirmation by the manufacturer of nonspecific

reactions after a lot of Blood ~roupingReagent has

been released should be reported promptly by the

manufacturer to the ~irector,Center for Biologics

Evaluation and Research.

 DRAFT

IV. AVIDITY TEST FOR SLIDE REAGENTS

A. REAGENT DILUTIONS

1. Prepare a 1 in 2 dilution of the reagent under test

by mixing equal parts of the reagent and AB serum,

group compatible serum, or a diluent approved by

the Director, Center for Biologics ~valuatfon and

Research.

1. Red blood cells should be prepared according to the

manufacturer's package insert.

C. MINIMUM TEST CELLS FOR AVIDITY

REAGENT RED BLOOD CELLS

Anti-D DCe (R,r)

dCce (rtr)

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

Cw positive (R,'r)

Anti-E dcEe (rVVr)

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

Anti-c dCce (rtr)

Anti-e dcEe (rHr)

Anti-CD Dce and dCce

(R,r and rtr)
*
rGr or rVVCr
Anti-DE Dce and dcEe

(Rr and rNr)

Anti-CDE Dce, dCce, and dcEe

(Ror,rtr, and r"r)
rCr *
* Only if the reagent is recommended for
detection of the G antigen by slide technique.
D. THE TEST (BY SLIDE METHOD)

1. The test is to be performed with both undiluted

reagent and the diluted reagent prepared in step

IVtA by the method recommended in the

manufacturer's package insert.

 DRAFT

E. INTERPRETATION OF THE TEST

1. Test results are observed and recorded at one half

of the manufacturer's recommended observation time

and at the end of the full recommended observation

time.

F. AVIDITY TESTING RESULTS ,

. .
-

1. Signs of agglutination should be observed with both

the undiluted and diluted reagent at one half of

the recommended observation time.

2. Clear macroscopic agglutination should be observed

with both the undiluted and diluted reagent at the

end of the recommended observation time and should

be reported as greater than or less than 1 mm.

V. TEST FOR PROZONE

A. REAGENT DILUTIONS
1. No dilution of the reagent under test is permitted.

B. RED BLOOD CELL PREPARATIONS

1. Obtain at least three red blood cell samples

representing three different Rh. phenotypes which

exhibit heterozygous or weak expression of the

antigen corresponding with the reagent antibody.

2. Fresh or frozen red blood cells may be used under

the following conditions:

a. Licensed reagent red blood cells may be used

an.y time before their expiration date.

b. Frozen red blood cells should have been frozen

within 7 days of collection from the donor and

should be used on the day of thawing.

c. All other red blood cell samples should be

used within 7 days of collection from the

donor.

3. Red blood cells should not be coated with

complement or immunoglobulin (should be direct

antiglobulin test negative).

4. Licensed reagent red blood cells may be used as

provided.

All other red blood cell samples should be washed

at least twice in isotonic saline or until a clear

 & . DRAFT

supernatant is obtained and then resuspended with

an approved diluent to the cell concentration

listed in the manufacturer's package insert.

C. CELLS SUGGESTED FOR USE IN THE TEST FOR PROZONE

. .
REAGENT RED BLOOD CELLS . -

DCce

(R,r1

DCcEe
( RlR2 )

~nti-E DCcEe

( RlR2 1

DCce and DCcEe

(R,r and RlR2)

DcEe and DCcEe

(R,r and R,R,)

D. THE TEST

1. For each cell sample to be tested label three

, and "60 minutestt
tubes, "15 minutestt,tt30minutestt
respectively.
2. Add the appropriate amount of the reagent under

test to all tubes.

If the manufacturer's package insert recommends the

use of * l drop of reagent, use 1 drop for this test.

If the manufacturer's package insert recommends the

use of 2 drops of reagent or 1 or 2 drops of

reagent, use 2 drops for this test.

3. Add 1 drop of each cell sample to its respective

tubes.

4. Mix and incubate for the time indicated on the tube

label according to the manufacturer's package

insert, i.e. at the temperature recommended for

those tests giving a negative result.

5. Centrifuge according to the package insert and

examine for agglutination. Grade the reactions as

in 1I.E.

 DRAFT

E, INTERPRETATION OF THE TEST

1, If the reaction grades are the sgme or increase as

the incubation time increases, no prozone is

present.

2. If the reaction grades decrease as the incubation

time increases, a prozone is present.

F. RESULTS

1. At least a 2+ reaction should be obtained with ALL

samples at ALL incubation times.

VI. TEST TO DETECT PROZONES - METHOD 2

A. REAGENT DILUTIONS

1. Prepare a 1+5 dilution of the reagent under test in

inert human serum (group AB or compatible with the

cells to be tested).

B. RED BLOOD CELL PREPARATIONS

1. See V.B.

C. CELLS SUGGESTED FOR USE IN THE TEST FOR PROZONE

1. See V.C.

D. TH.E TEST

The 1+5 dilution and undiluted reagent will be tested in

parallel.

1. For each cell to be tested, label two sets of

tubes, nI.S.w, "1 minuten, " 3 minutes", "5
minutesu, and "10 minutes".
2. Add the appropriate amount of the reagent under

test to all tubes.

If the manufacturerfs package insert recommends the

use of 1 drop of reagent, use 1 drop for this test.

If the manufacturerfs package insert recommends the

use of 2 drops of reagent or 1 or 2 drops of

reagent, use 2 drops for this test.

3. Add 1 drop of each cell sample to its respective

tubes.

4. Mix and incubate at room temperature for the time

indicated on the tube label.

 DRAFT

5. Centrifuge according to the package insert and

examine for agglutination. Grade the reactions as

in 1I.E.

E. INTERPRETATION OF THE TEST -

1. If the reaction grades with the diluted reagent are

stronger than the reaction grades with the

undiluted reagent, a prozone is present.

2. If the reaction grades with the diluted reagent are

equal to or weaker than the reaction grades with

the undiluted reagent, no prozone is present.

F. RESULTS

1. The reagent should not exhibit any prozone.

 -- .--.- -.

DRAFT

LOW PROTEIN RH BLOOD GROUPING REAGENTS

GENERAL INFORMATION

These recommended methods are provided to help assist manufac:turers

in pursuing new product license applications and amendments to

existing product license applications. The methods described

herein do not bind the agency, and manufacturers may consitler use

of other methods. In cases where manufacturers wish to use methods

other than those described herein, FDA recommends that the matter

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

values, specificity results, and other matters referred to in this

document are intended to assist manufacturers in preparing

submissions to FDA. The information is based on current knowledge

and is not meant to be all inclusive and should not be viewed as

ensuring approval or the only means of achieving FDA approval.

Following the methods provided in this document will assist in the

approval process, but does not guarantee approval. FDA will review

applications on an individual basis and approvals will be qranted

when supported by scientific evidence.

I. REFERENCE PREPARATIONS

A. The Reference Blood Grouping Reagents listed below can be

obtained from:

Center for Biologics Evaluation and Research

Food and Drug Administration
8 8 0 0 Rockville Pike
Bethesda, MD. 2 0 8 9 2
USA
Anti-CD for evaluation of IgM, Anti-D

products

Anti-C for saline tube test

Anti-E for saline tube test

NOTE : FDA Reference Blood Grouping Reagents are not

routinely available to anyone except U.S. licensed
manufacturers and amounts issued will be
proportional to lots released in the previous year.
For Blood Grouping Reagents for which there is no

Reference Blood Grouping Reagent, it is strongly

recommended that a previously approved lot of

reagent be used as an in-house control reaglent.

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

accompanying package insert only for determining the

potency of Blood Grouping Reagents as part of their final

lot release testing.

In-house reference materials should be developed for all

stability testing, in process testing or proauct

development purposes.

22. POTENCY TESTING

A. REAGENT DILUTIONS

1. Beginning with the undiluted reagent, prepare

separate master two-fold serial dilutions (1 in 2,

1 in 4, etc.) of the test reagent using isotonic

saline containing 1-2% bovine albumin or another

diluent approved by the Direckor, Center for

Biologics Evaluation and Research. Test tubes

should be of a size that facilitates adequate

mixing of the contents (12 X 75 mm or larger).

If the endpoint is expected to exceed 1024,

accuracy will be improved if direct intermediate

dilutions are done to keep the number of serial

transfers to less than 10. (e.g., If the expected

endpoint is 4096, prepare an initial 1:10 dilution

with the same diluent as used above.)

NOTE : All titrations should be carried to a

negative endpoint. (See E.4)

2. Prepare master dilutions of the Reference Blood

Grouping Reagent(s) or in-house control reagent as

in paragraph 1 of this section. For test reagents

containing multiple antibodies (ex. Anti-CDE),

dilutions of each of the corresponding Reference

Blood Grouping Reagents should be made separately.

3. A separate, clean pipet or pipet tip should be used

for each dilution (including any intermediate
dilutions) to avoid carryover of higher reagent
concentrations.
4. The last tube should contain diluent only and serve

as a diluent control.

B. RED BLOOD CELL PREPARATIONS

Fresh or frozen red blood cells may be used for preparing

cell suspensions for the potency testing of all Blood

Grouping Reagents under the following conditions:

1. Red blood cells of any age may be used, provided

the titer values of the Reference Blood '~rouping

Reagent(s) or the in-house control reagent are

within an acceptable range.

2. Red blood cells may be frozen and thawed for use in

the preparation of cell suspensions for reagent

evaluation. To ensure that the correct cell has

been thawed, appropriate controls should be used to

demonstrate the desired reactivity and identity of

the thawed red blood cells on the day of use.

The method of freezing, storing, and thawing red

blood cells, including a description of the

cryoprotective medium should be described in detail

and should be approved by the Director, Center for

Biologics Evaluation and Research as a license

amendment before use in control testing.

3. Each batch of red blood cells for use in con~trol

testing of reagents requiring indirect antiglot~ulin

technique should be tested for absence of

complement or immunoglobulin molecules (DAT

negative) on the day of use.

4. Red blood cells should be washed at least-twice in

isotonic saline or until a clear supernate is

obtained and then resuspended to a 2% suspension in

isotonic saline containing 1-2% bovine albumin or

another diluent approved by the Direc-tor, Center

for Biologics Evaluation and Research.

C. MINIMUM TEST CELLS FOR POTENCY

REAGENT RED BLOOD CELLS

Anti-D Dce

( Ror

Anti-C dCce

(r'r)

Anti-E dcEe

(rttr

Anti-c DCcEe

( RIR2

Anti-e dcEe

(rltr)

Anti-CD dCce and Dce

(r'r and Ror)

Anti-DE Dce and dcEe

(Ror and rl1r)

Anti-CDE dCce and Dce and dcEe

(rlr and &r and rwr)

D. THE TEST (BY TUBE METHOD)

1. Place 0.1 milliliter of each reagent dilution in a

separate, clean test tube (approximately 10 X 7 5 mm

or 1 2 X 7 5 mm).

2. If a Reference Blood Grouping Reagent is available,

place 0.1 milliliter of each Reference Blood
Grouping Reagent dilution in a separate, clean test
tube (approximately 10 X 7 5 rnm or 12 X 7 5 mm).
3. Add 0.1 milliliter of the appropriate 2% cell

suspension to each test tube.

4. Mix the contents of each tube thoroughly and

'incubate the test tubes at 3 7 C for 15 minutes.

If no Reference Blood Grouping Reagent is

available, incubate at 3 7 C for the shortest period

of time recommended in the manufacturer's package

insert.

5. Centrifuge for 1 minute at approximately 1000 rpm

(100-125 rcf) or 15 seconds at approximately.3400

30

 DRAFT

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.

 2. Products of polyclonal origin which are recommencled

for tube test methods for which there are no

Reference Blood ~roupingReagents available should

have a potency titer value of at least a 1+

reaction with a 1:4 dilution of reagent:

eg. Anti-c (saline) , -

Anti-e (saline)
3. Products of monoclonal origin which are recommendied

for tube test methods for which there are no

Reference Blood Grouping Reagents available shou,ld

have a potency titer value of at least a 1+

reaction with a 1:8 dilution of reagent:

eg. Anti-c (saline)

Anti-e (saline)

Manufacturers that wish to establish potency titer

values other than these are to obtain approval from

the Director, Center for ~iologicsEvaluation and

Research at the time of license application.

4. Products recommended for use in automated or

microplate systems without user dilution ( as
supplied) should be sufficiently potent that a two-
fold dilution prepared with an approved diluent
will produce the same qualitative test result as
the undiluted product when tested in accordance
with the manufacturer's package insert.
111. SPECIFICITY TESTING

A. REAGENT DILUTIONS

1. No dilution of the reagent under test is permitted.

B. RED BLOOD CELL PREPARATIONS

Fresh or frozen red blood cells may be used for preparing

cell suspensions for the specificity testing of all Blood

Grouping Reagents under the following conditions:

1. Any cells of any age may be used in the '!Test to

Confirm Reactivity with Antigen Positive Cells"

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

Contaminating Antibodiesgg (III.C.2) licensed

reagent red blood cells may be used any time before

their expiration date. All other red blood cell

samples should be used within 7 days of collection

from the donor.

 DRAFT

Manufacturers that wish to use cells more than 7

days after collection from the donor are to obtain

approval from the Director, Center. for Biologics

Evaluation and Research, and are to provide

sufficient data to support the request.

2. Red blood cells meeting the criteria of paqagraph 1

of this section may be frozen and thawed $or use in
the preparation of cell suspensions for reagent
evaluation. To ensure that the correct ce.11 has
been thawed, appropriate controls should be used to
demonstrate the desired reactivity and identity of
the thawed red blood cells on the day of use. In
the case of cells expressing low frelquency
antigens, testing for several common antigens may
serve to adequately identify the cell.
The method of freezing, storing, and thawing red

blood cells, including a description of the

cryoprotective medium should be described in detail

and should be approved by the Director, Center for

Biologics Evaluation and Research before use in

control testing of licensed reagents.

3. Each batch of red blood cells for use 'in control

testing of reagents requiring indirect antiglobulin
technique should be tested for absence of
complement or immunoglobulin molecules (DAT
negative) on the day of use. .

4. Licensed reagent red blood cells may be used as

provided.

All other red blood cell samples should be washed

at least twice in isotonic saline or until a clear

supernatant is obtained and then resuspended with

an approved diluent to the cell concentration

listed in the manufacturer's package insert.

C. MINIMUM TESTING FOR SPECIFICITY

1. TEST TO CONFIRM REACTIVITY WITH ANTIGEN PO:~ITIVE

CELLS

a. At least 4 different donors whose red blood

cells exhibit weak or heterozygous expression

of the antigen should be tested.

 DRAFT

b. When testing reagents containing multiple

antibodies, the reactivity of each specificity

should be confirmed separately by using 4

different red blood cells possessing only one

of the antigens for each different

specificity.

ex. For Anti-CDE reagents, at least four

donors should be used to confirm .the

reactivity of the Anti-C component, at least

four donors should be used to confirm 'the

reactivity of the Anti-D component, and at

least four donors should be used to confirm

the reactivity of the Anti-E component.

c. Include at least one red blood cell which does

not exhibit the expression of the antigen as a

negative control.

2. TEST TO CONFIRM ABSENCE OF CONTAMINATING ANTIBODIES

Test the reagent for the presence of antibodies

corresponding to the following antigens by one of

the methods listed below.

A, BI H I Lea, Leb, I,. K, k, Kpa, Kpb, Jsb, P,, D, C,

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

Fyb, Xga, Doa, Dob, Yta, Ytb, Lan, Coat Cob, M9, 91ra,

and Sda.

If the source material is a monoclonal antib'ody

from a previously characterized and licensed clone,

this list may be shortened as follows:

A, B , H I Lea,Leb, I, K, k, Kpb, Jsb, PI, D, C, E, C,

e, MI N, S, s, U, Lub,Jka, Jkb, Fya, and Fyb.
Approval for the use of fewer antigens than

included on this list may be requested from the

Director, Center for Biologics Evaluation and

Research by a manufacturer at the time of

submission of the first protocol.

a. Perform a direct test for the presence of

contaminating antibodies by using red blood
cells from at least 4 different donors whose
cells lack the antigen corresponding to the
reagent antibody.
 DRAFT

b. When red blood cells lacking the antigen

corresponding to the reagent antibody under

test are not available, the reagent antibody

may be adsorbed to exhaustion with cells of a

known phenotype.

The adsorbed serum may then be tested against

red blood cells which exhibit any. antigens

which were not present on the cells used for

adsorption. The methods for adsorption and

subsequent testing should be approved by the

Director, Center for Biologics Evaluatioln and

Research.

c. When direct tests are impractical, the

Director, Center for Biologics Evaluation and

Research may approve procedures whereby

antibodies may be presumptively excluded by

testing an appropriate number of non-reactive

red blood cell samples to provide statistical

assurance of the absence of contaminating

antibodies.

d. Red blood cell samples from four different

donors may be used to confirm presumptively

the absence of contaminating antibodies to

antigens having an incidence of greater than

99% in the general population of the United

States.

3. TEST TO CONFIRM ABSENCE OF ANTI-A AND ANTI-B

a. Group A, and B red blood cells lacking the

antigen corresponding to the reagent antibody

should be tested. Group A,B red blood cells

may be substituted for A, and/or B red blood

cells if either are unavailable.

Adsorbed serum may be used as in III.C.2.b

above.

4. PHENOTYPES RECOMMENDED FOR TESTING

As a minimum, red blood cells exhibiting the

following phenotypes should be used in the

specificity testing outlined in steps 1, 2, and 3

above.

REAGENT RED BLOOD' C E L L S

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

( R , r , R o r , r l r , and r I 1 r )

A, dce, B dce, a n d 0 dce ( r r )

Vw p o s i t i v e . -

.
3 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
Rh phenotypes and reactive by
Indirect A n t i g l o b u l i n T e s t only *
Anti-D C a t e g o r y I V , V, and V I c e l l s
(monoclonal)

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 . RzR2 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 DCEe 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 DCe (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,)

DCcEe f neg (RlR2)

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 wo r r W r )

A, dce, B dce, and 0 dce ( r r )

rGro 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 or 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 )

rGr***
 DRAFT

* For Anti-D reagents recommended for D" testing.

** r'"r cells may be used in addition to but not

as a substitute for C+ Ce- cells
*** Recommended if labeling indicates detection of
G antigen . .
. . - .

D. THE TESTS

1. To confirm reactivity with antigen positive cells,

each lot of Blood Grouping Reagent should be tested

and results interpreted by all test methods

described in the manufacturer's package insert.

Minimum parameters (drops of reagent, inculbation

time, centrifugation, etc.) should be followced.

2. To confirm absence of contaminating antibodies,

each lot of Blood Grouping Reagent should be tested
and results interpreted by the most sensitive test
method(s) described in the manufacturer's package
insert. Maximum parameters (drops of reagent,
incubation time, centrifugation, etc.) shou~ld be
followed.
E. SPECIFICITY RESULTS

1. No hemolysis or rouleaux formation should be

detected by any of the methods in the

manufacturer's package insert.

2. Red blood cells which exhibit the antigen

corresponding to the reagent antibody should yield

at least a 2+ reaction. If any of the four samples

tested yields less than a 2+ reaction, red blood

cells from four additional donors who exhibit the

antigen should be tested. The test is considered

satisfactory if no more than one of eight red blood

cell samples yields less than a 2+ reaction with

the test reagent.

When testing unusual phenotypes, other criteria for

reactivity may apply. For example, a llarger

percentage of C+ Ce- red blood cells may not yield

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

clearly positive macroscopic result.

3. The negative control cell(s) in step 1II.C. 1 should

yield a negative reaction by each test method

described in the manufacturer's package insert.

 4. Tests with red blood cells which lack the antigen

corresponding to the reagent antibody and tests

with adsorbed reagent should be negative, thus

confirming the absence of significant contaminating

antibodies directed at the antigens listed in

III.C.2

5. The manufacturer should list on the lot release

protocol and in the "Specific Performance

Characteristics" section of the package insert

those red blood cell antigens listed in I11 .C.2 for

which no specificity tests have been performed.

If desired, the red blood cell phenotype of the

antibody donor(s) may also be listed as presumptive

evidence that antibodies to those factors are not

present.

6. Tests with group A, and B red blood cells should :be

negative, thus confirming the absence of anti-A and

anti-B.

7. Confirmation by the manufacturer of nonspecific

reactions after a lot of Blood Grouping Reagent htas

been released should be reported promptly by tlhe

manufacturer to the Director, Center for Biologilcs

Evaluation and Research.

AVIDITY TEST FOR SLIDE REAGENTS

A. REAGENT DILUTIONS

1. Prepare a 1 in 2 dilution of the reagent under test

by mixing,equal parts of the reagent and AB seruim,

group compatible serum, or a diluent approved Iby

the Director, Center for Biologics Evaluation and

Research.

B. RED BLOOD CELL PREPARATIONS

1. Red blood cells should be prepared according to tlhe

manufacturer's package insert.

 DRAFT

C. MINIMUM TEST CELLS FOR AVIDITY

REAGENT RED BLOOD CELLS

Anti-D DCe (R,r)

Anti-C dCce (rfr) . -

C+ Ce- (R,R, or R,)
Cw positive (RlWr)
dcEe (rttr)
E+ CE- (R,R, or R,r )
dCce (rfr)

dcEe (rttr)

Dce and dCce

(R,r and rfr)

*
rCr or rttCr
Dce and dcEe

( R r and rmr)

Dce, dCce, and dcEe

(Rorfr f r f and rttr)
rGr *
* Only if the reagent is recommended for
detection of the G antigen by slide technique.
D. THE TEST (BY SLIDE METHOD)

1. The test is to be performed with both undiluted

reagent and the diluted reagent prepared in step

IV,A by the method recommended in the

manufacturerfs package insert.

E. INTERPRETATION OF THE TEST

1. Test results are observed and recorded at one half

of the manufacturer's recommended observation time

and at the end of the full recommended observation

time.

F. AVIDITY TESTING RESULTS

1. Signs of agglutination should be observed with both

the undiluted and diluted reagent at one half of
the manufacturerfs recommended observation time.
2. Clear macroscopic agglutination should be observed

with both the undiluted and diluted reagent at the

 end of the manufacturer's recommended observati.on

time and should be reported as greater than or less

mm.

than 1
V. TEST FOR SPONTANEOUS AGGLUTINATION

A. REAGENT DILUTIONS .., -

. .

1. No dilution of the reagent under test is permitted.

B. RED BLOOD CELL PREPARATION

1. Obtain c positive group 0 red blood cells from one

donor. (When testing an Anti-c reagent, use Rh(D)

positive group 0 cells.)

2. Coat the red blood cells heavily with an IgG anti-c

(anti-D when testing an Anti-c reagent) such that. a
3-4+ direct antiglobulin test (DAT) is achieved and
positive reactions are obtained with a high protein
Rh control reagent, but negative reactions are
obtained with a saline control.
The exact procedure for coating the red .blood cells

will depend on the specific antibody chosen for

coating and its strength.

C. THE TEST

1. Test the coated cell sample according to the

manufacturer's package insert.

D. INTERPRETATION

1. Blood Grouping Reagents for use by the saline tube

test method should not spontaneously agglutinate

immunoglobulin coated red blood cells.

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

group results should be recommended or supplied.

VI. TEST FOR PROZONE

A. REAGENT DILUTIONS

1. No dilution of the reagent under test is permitted.

 DRAFT

B. RED BLOOD CELL PREPARATIONS

1. Obtain at least three red blood cell samples

representing three different Rh phenotypes which

exhibit heterozygous or weak expression of the

antigen corresponding with the reagent antibody.

2. Fresh or frozen red blood cells may be used under

the following conditions:
a. Licensed reagent red blood cells may be used

any time before their expiration date.

b. Frozen red blood cells should have been f'rozen

within 7 days of collection from the donor and

should be used on the day of thawing.

c. All other red blood cell samples should be

used within 7 days of collection from the

donor.

3. Red blood cells should not be coated with

complement or immunoglobulin ( should be diirect
antiglobulin test negative).
4. Licensed reagent red blood cells may be usled as

provided.

All other red blood cell samples should be washed

at least twice in isotonic saline or until a clear

supernatant is obtained and then resuspended with

an approved diluent to the cell concentration

listed in the manufacturer's package insert.

C. CELLS SUGGESTED FOR USE IN THE TEST FOR PROZONE

REAGENT RED BLOOD CELLS

DCce
( R1r

Anti-C DCcEe

( RlR2 )

DCcEe
( RlR2 )

DCce and DCcEe

(R,r and R,R,)

DcEe and DCcEe

(R,r and R,R2)

 D. THE TEST

1. For each cell sample to be tested label three

tubes, "15 minutestt,"30 minutestt,and "60 minutestt

respectively.

2. Add the appropriate amount of the reagent under

test to all tubes.

If the manufacturer's package insert recommends the

use of 1 drop of reagent, use 1 drop for this test.

If the manufacturer's package insert recommends the

use of 2 drops of reagent or 1 or 2 drops of

reagent, use 2 drops for this test.

3. Add 1 drop of each cell sample to its respective

tubes.

4. Mix and incubate for the time indicated on the tube

label according to the manufacturer's package

insert, i.e. at the temperature recommended1 for

those tests giving a negative result.

5. Centrifuge according to the package insert. and

examine for agglutination. Grade the reactio:nsas

in 1I.E.

1. If the reaction grades are the same or increase as

the incubation time increases, no prozone is

present.

2. If the reaction grades decrease as the incubation

time increases, a prozone is present.

F. RESULTS

1. At least a 2+ reaction should be obtained with ALL

samples at ALL incubation times.

VII. TEST TO DETECT PROZONES - METHOD 2

A. REAGENT DILUTIONS

1. Prepare a 1+5 dilution of the reagent under test in

inert human serum (group AB or compatible with the

cells to be tested.)

B. RED BLOOD CELL PREPARATIONS

1. See V1.B.

 DRAFT

C. CELLS SUGGESTED FOR USE IN THE TEST FOR PROZONE

1. See VI .C.

D. THE TEST

The 1+5 dilution and undiluted reagent will be .tested in

*
parallel.

1. For each cell to be tested, label two sets of

tubes, "1. S. It, minutett, " 3 minutestt, "5
minutesn, and "10 minutes".
2. Add the appropriate amount of the reagent under

test to all tubes.

If the manufacturer's package insert recommends the

use of 1 drop of reagent, use 1 drop for this test,

If the manufacturer's package insert recommends the

use of 2 drops of reagent or 1 or 2 drops of

reagent, use 2 drops for this test.

3. Add 1 drop of each cell sample to its respective

tubes.
4. Mix and incubate at room temperature for the time

indicated on the tube label.

5,. Centrifuge according to the package insert and

examine for agglutination. Grade the reactions as

in 1I.E.

1. If the reaction grades with the diluted reagent are

stronger than the reaction grades with the

undiluted reagent, a prozone is present.

2. If the reaction grades with the diluted reagent are

equal to or weaker than the reaction grades with
the undiluted reagent, no prozone is present.
F. RESULTS

1. The reagent should not exhibit any prozone.

 DRAFT

RARE BLOOD GROUPING REAGENTS

GENERAL INFORMATION

These recommended methods are provided to help assist manuf aciturers

in pursuing new product license applications and amendments to

existing product license applications. The methods described

herein do not bind the agency, and manufacturers may considler use

of other methods. In cases where manufacturers wish to use methods

other than those described herein, FDA recommends that the matter

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

values, specificity results, and other matters referred to i.n this

document are intended to assist manufacturers in preparing

submissions to FDA. The information is based on current knotwledge

and is not meant to be all inclusive and should not be viewed as

ensuring approval or the only means of achieving FDA approval.

Following the methods provided in this document will assist in the

approval process, but does not guarantee approval. FDA will review

applications on an individual basis and approvals will be qrranted

when supported by scientific evidence.

I. REFERENCE PREPARATIONS

A. There are no Reference Blood Grouping Reagents ava.ilable

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

strongly recommended that a previously approved lot of

reagent be used as an in-house control reagent.

I POTENCY TESTING

A. REAGENT DILUTIONS

1. Beginning with the undiluted reagent, prepare

separate master two-fold serial dilutions (1. in 2,
1 in 4, etc.) of the test reagent using isotonic
saline containing 1-2% bovine albumin or amother
diluent approved by the Director, Center for
Biologics Evaluation and Research. Test tubes
should be of a size that facilitates adequate
mixing of the contents (12 X 75 mm or larger).
If the endpoint is expected to exceed 1024,

accuracy will be improved if direct intern~ediate

dilutions are done to keep the number of serial

transfers to less than 10. (e.g., If the expected

endpoint is 4096, prepare an initial 1:10 dilution

with the same diluent as used above.)

NOTE : All titrations should be carried to a

negative endpoint. (See E.4)
2- Prepare master dilutions of the in-house control
reaqent as in paragraph 1 of this section.
 DRAFT

3. A separate, clean pipet or pipet tip should be used

for each dilution (including any intermediate
dilutions) to avoid carryover of higher reaqent
concentrations.
4. The last tube should contain diluent only and serve
as a diluent control.
B. RED BLOOD CELL PREPARATIONS

Fresh or frozen red blood cells may be used for preparing

cell suspensions for the potency testing of all Blood

Grouping Reagents under the following conditions:

1. Red blood cells of any age may be used, provided

the titer values of the in-house control reagent

are within an acceptable range.

Red blood cells may be frozen and thawed for use in

the preparation of cell suspensions for reaqent

evaluation. To ensure that the correct cell has

been thawed, appropriate controls should be used to

demonstrate the desired reactivity and identity of

the thawed red blood cells on the day of use. In

the case of cells expressing low. frequency

antigens, testing for several common antigens may

serve to adequately identify the cell.

The method of freezing, storing, and thawing red

blood cells, including a description of the

cryoprotective medium should be described in detail

and should be approved by the Director, Center for

Biologics Evaluation and Research as a license

amendment before use in control testing of reagent.

3. Each batch of red blood cells for use in control

testing of reagents requiring indirect antiglobulin

technique should be tested for absence of

complement or immunoglobulin molecules (DAT

negative) on the day of use.

4. Red blood cells should be washed at least twice in

isotonic saline or until a clear supernate is

obtained and then resuspended to a 2% suspension in

isotonic saline containing 1-2% bovine albumin or

another diluent approved by the Director, Center

for Biologics Evaluation and Research.

C. MINIMUM TEST CELLS FOR POTENCY

1. At least 2 different donors with phenotypes

exhibiting weak and/or heterozygous expression of -
the antigen, where applicable.
For example, Anti-Lea and Anti-Leb are excluded.

 DRAFT

D. THE TEST (BY TUBE METHOD)

1. Place 0.1 milliliter of each reagent dilution in a

separate, clean test tube (approximately 10 X 75 mm

or 12 X 75 mm).

2. Add 0.1 milliliter of the appropriate,--,2%cell

suspension to each test tube.

3. Mix the contents of each tube thoroughly and

incubate the test tubes for the shortest incubation

time at the temperature recommended in the

manufacturer's package insert for the product. For

rapid tube reagents incubate at room temperature

(RT; 20-30 C) for 5 minutes.

4. Centrifuge at the lowest speed and for the shlortest

period of time recommended in the manufacturer's

package insert.

5. Perform the indirect antiglobulin test according to

the manufacturer's package insert, if recommended.
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 clumps.

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 granules, but

not definite small clumps. Results should be

recorded as doubtful. For purposes o:E this

paragraph, doubtful reactions are deemed to be

negative.

0
Negative reaction- cell button dislodges into

no visible clumps.

 DRAFT

3. The p o t e n c y t i t e r v a l u e is t h e r e c i p r o c a 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 which t h e r e a c t i o n i s
g r a d e d a t l+.
The d i l u t i o n c a u s e d by t h e a d d i t i o n o f t h e r e d
blood cells should not be considered as
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 e a g e n t .
. -
4. T e s t r e s u l t s s h o u l d show a t l e a s t o n e t u b e w i t h n o
agglutination a f t e r t h e endpoint. 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 T I T E R VALUES
1. P r o d u c t s of p o l y c l o n a l o r i g i n which a r e recomnlended
f o r t u b e t e s t methods s h o u l d h a v e an a v e r a g e
potency t i t e r v a l u e a s follows:
a. 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 of
reagent :
Anti-K
Anti-k
Anti-Jka
A n t i -Fya
Anti-Cw
b. A 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
reagent :

c. A 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
Anti-Jsa
Anti-Jsb
Anti-Fyb
Anti-N
Anti-Lea
Anti-Leb
Anti-Dia
Ant i - M q

Anti-Jkb

Anti-Xga

Anti-Cob

Anti-Wr"

 DRAFT

2. Products of monoclonal origin which are recommended

for tube test methods should have an average

potency titer value of at least 1+ with a 1:8

dilution of reagent.

Manufacturers that wish to establish potency titer

values other than these are to obtain approval from

the Director, Center for Biologics Evaluation and

Research at the time of license application.

3. Products recommended for use in automated or

microplate systems without user dilution (as

supplied) should be sufficiently potent that a two-

fold dilution prepared with an approved diluent

will produce the same qualitative test result as

the undiluted product when tested in accordance

with the manufacturer's package insert.

111. SPECIFICITY TESTING

A. REAGENT DILUTIONS

1. No dilution of the reagent under test is permitted.

B. RED BLOOD CELL PREPARATIONS

Fresh or frozen red blood cells may be used for preparing

cell suspensions for the specificity testing of all Blood

Grouping Reagents under the following conditions:

1. Any cells of any age may be used in the "Test to

Confirm Reactivity with Antigen Positive Cellsvv

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

Contaqinating Antibodies" (III.C.2) licensed

reagent red blood cells may be used any time before

their expiration date. All other red blood cell

samples should be used within 7 days of collection

from the donor.

Manufacturers that wish to use cells more than 7

days after collection from the donor are to obtain

approval from the Director, Center for Biol.ogics

Evaluation and Research, and are to provide

sufficient data to support the request.

2. Red blood cells meeting the criteria of paragraph 1

of this section may be frozen and thawed for use in

the preparation of cell suspensions for reagent

evaluation. To ensure that the correct cell has

been thawed, appropriate controls should be used to

demonstrate the desired reactivity and identity of

the thawed red blood cells on the day of use, In

the case of cells expressing low frequency

antigens, testing for several common antigens may

serve to adequately identify the cell.

 DRAFT

The method of freezing, storing, and thawing red

blood cells, including a description of the

cryoprotective medium should be described in detail

and should be approved by the Director, Center for

~iologics Evaluation and Research before use in

control testing of licensed reagents.

3. Each batch of red blood cells for use in' c!ontrol

testing of reagents requiring indirect antiglobulin
technique should be tested for absence of
complement or immunoglobulin molecules (DAT
negative) on the day of use.
4. Licensed reagent red blood cells may be used as

provided.

All other red blood cell samples should be washed

at least twice in isotonic saline or until a clear

supernatant is obtained and then resuspended with

an approved diluent to the cell concentration

listed in the manufacturer's package insert.

I
C. MINIMUM TESTING FOR SPECIFICITY I

1. TEST TO CONFIRM REACTIVITY WITH ANTIGEN POSITIVE

CELLS

a. At least 4 different donors whose red blood

cells exhibit weak or heterozygous expression

of the antigen should be tested.

b. When testing reagents containing multiple

antibodies, the reactivity of each specificity

should be confirmed separately by using 4

different cells possessing only one of the

antigens for each different specificity.

c. Include at least one red blood cell which does

not exhibit the expression of the antigen as a

negative control.

2. TEST TO CONFIRM ABSENCE OF CONTAMINATING ANTIBODIES

Test the reagent for the presence of antibodies

corresponding to the following antigens by one of

the methods listed below.

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",

and Sd".

 DRAFT

If the source material is a monoclonal antibody

from a previously characterized and licensed clone,

this list may be shortened as follows:

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

e, M I N I S, s, U, Lub, Jk", Jkb, Fy", and Fyb
Approval for the use of fewer antigens than

included on this list may be requested from the

Director, Center for Biologics Evaluation and

Research by a manufacturer at the time of

submissi/on of the first protocol.

a. Perform a direct test for the presence of

contaminating antibodies by using red blood

cells from at least 4 different donors whose

cells lack the antigen corresponding to the

reagent antibody.

When red blood cells lacking the antigen

corresponding to the reagent antibody under

test are not available, the reagent antibody

may be adsorbed to exhaustion with cells of a

known phenotype.

The adsorbed serum may then be tested against

red blood cells which exhibit any antigens

which were not present on the cells used for

adsorption. The methods for adsorption and

subsequent testing should be approved by the

Director, Center for Biologics Evaluation and

Research.

.When direct tests are impractical, the

Director, Center for Biologics Evaluation and

Research may approve procedures whereby

antibodies may be presumptively exclutded by

testing an appropriate number of non-reactive

red blood cell samples to provide statistical

assurance of the absence of contaminating

antibodies.

Red blood cell samples from four different

donors may be used to confirm presumptively

the absence of contaminating antibodies to

antigens having an incidence of greater than

99% in the general population of the United

States.

 DRAFT

3. TEST TO CONFIRM ABSENCE OF ANTI-A AND ANTI-B

a. Group A, and B red blood cells lacking the

antigen corresponding to the reagent antibody

should be tested. Group A,B red blood cells

may be substituted for A, and/or B red blood

cells if either are unavailable.

Adsorbed serum may be used as in III.C.2.b

abo.ve .
4. ADDITIONAL PHENOTYPES RECOMMENDED FOR TESTING

REAGENT RED BLOOD CELLS

Anti-A, A,, A,, A,B, A,B, B, and 0

Anti-Leb 6 different A, and/or A,B Le(b-I) *

Anti-P, At least 2 P, weak (as determined by

titration studies)

* Group A and AB cells which do react with anti-A,

and do not react with anti-'H.
THE TESTS

1. To confirm reactivity with antigen positive cells,

each lot of Blood Grouping Reagent should be tested

and results interpreted by all test methods

described in the manufacturer's package insert.

Minimum parameters (drops of reagent, incubation

time, centrifugation, etc.) should be followed.

2. To confirm absence of contaminating antibodies,

each lot of Blood Grouping Reagent should be tested
and results interpreted by the most sensitive test
method(s) described in the manufacturer's package
insert. Maximum parameters (drops of reingent,
incubation time, centrifugation, etc. ) shou~ld be
followed.
 DRAFT

E. SPECIFICITY RESULTS

1. No hemolysis or rouleaux formation should be

detected by any of the methods in the

manufacturer's package insert.

Red blood cells which exhibit the--=antigen

corresponding to the reagent antibody should yield

at least a 2+ reaction. If any of the four samples

tested yields less than a 2+ reaction, red blood

cells from four additional donors who exhibit the

antigen should be tested. The test is considered

satisfactory if no more than one of eight red blood

cell samples yields less than a 2+ reaction with

the test reagent.

When testing unusual phenotypes, other criteria for

reactivity may apply. For example, Fyx red blood

cells may not yield a 2+ reaction with Anti-Fyb but

should yield a clearly positive macroscopic result.

3. Thenegativecontrol cell(s) instepIII.C.1 should

yield a negative reaction by each test nnethod

described in the manufacturer's package insert.

4. Tests with red blood cells which lack the antigen

corresponding to the reagent antibody and tests

with adsorbed reagent should be negative, thus

confirming the absence of significant contaminating

antibodies directed at the antigens listed in

III.C.2

5. The manufacturer should list on the lot release

protocol and in the "Specific Performance

Characteristics*' section of the package insert

those red blood cell antigens listed in III.C.2 for

which no specificity tests have been performed.

If desired, the red blood cell phenotype of the

antibody honor(s) may also be listed as presunnptive

evidence that antibodies to those factors are not

present.

6. Tests with group A, and B red blood cells should be

negative, thus confirming the absence of anti--Aand
anti-B.
7. Confirmation by the manufacturer of nonspc'cific

reactions after a lot of Blood Grouping Reagent has

been released should be reported promptly by the

manufacturer to the Director, Center for Biologics

Evaluation and Research.

 DRAFT

IV. AVIDITY TEST FOR SLIDE REAGENTS

A. REAGENT DILUTIONS

1. Prepare a 1 in 2 dilution of the reagent under test

by mixing equal parts of the reagent and AB serum,

group compatible serum, or a diluent approved by

the Director, Center for ~iologics valuation and

Research.

B. RED BLOOD CELL PREPARATIONS

1. Red blood cells should be prepared according to the

manufacturer's package insert.

C. MINIMUM TEST CELLS FOR AVIDITY

1. Red blood cells from at least two different donors

exhibiting weak and/or heterozygous expression of

the antigen corresponding to the reagent antibody

should be used.

D. THE TEST (BY SLIDE METHOD)

1. The test is to be performed with both undiluted

reagent and the diluted reagent prepared in step

IV,A by the method recommended in the

manufacturer's package insert.

E. INTERPRETATION OF THE TEST

1. Test results are observed and recorded at one half

of the manufacturer's recommended observation time
and af the end of the full recommended observation
time .
F. AVIDITY TESTING RESULTS
1. Signs of agglutination should be observed with both

the undiluted and diluted reagent at one half of

the manufacturer's recommended observation time.

2. Clear macroscopic agglutination should be observed

with both the undiluted and diluted reagent at the
end of the manufacturer's recommended observation
time and should be reported as greater than or less
than 1 mm.