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BS/90.1645

WORLD HEALTH ORGANIZATION

ORGANISATION MONDIALE DE LA SANTE

ENGLISH ONLYEXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATIONGeneva 16 to 23 October 1990

A COLLABORATIVE INVESTIGATION ON ANTI-D (ANTI-RHO) COMPLETEBLOOD-TYPING SERUM (CHEMICALLY MODIFIEW,

by Drs W.J. Duimel and H.W. KrijnenCentral Laboratory of the Netherlands Red Cross

Blood Transfusion ServiceAmsterdam

INTRODUCTION

In the meeting in 1985 the Expert Committee on Biological Standardization notedthat the Central Laboratory of the Netherlands Red Cross had been unsuccessful inacquiring a complete anti-D blood-typing serum that showed satisfactory potency andstability in accelerated degradation tests. The Committee requested the CentralLaboratory to prepare and examine a chemically modified anti-D complete blood-typingserum.

This report gives the results of the preparation and the stability test of aproposed standard, and the results of a collaborative study to test its suitabilityfor use as an International Standard for anti-D (anti-Rho) complete blood-typingserum (chemically modified).

MATERIALS AND METHODS

1500 mJ_s of a chemically modified hyperimmune anti-D serum of human origin wasobtained from Gammachim, Zurich, Switzerland. To improve stability and to allow forfreeze-drying, the anti-D serum was mixed with 105 mls of Bovine

Serum Albumin 30%, especially made for serological use (Organon Teknika, TurnhoutBelgium). The mixture was heat-inactivated for 30 minutes at 56°C and then

filtered through a presterilized 0.22 um membrane filter.

The mixture was distributed in precisely measured equal volumes of 1.0 ml intoall-glass ampoules, freeze-dried, filled with nitrogen and sealed by glass fusion.

The, dried content of the ampoules was determined by weighing 24 randomlyselected ampoules with and without the dried serum.

The water content of the ampoules was determined by the Karl Fischer method(USP)

Accelerated degradation tests were done at three different temperatures, theampoules being stored at those temperatures for various periods of time.

Specificity tests of the proposed standard were done with cells of differentABO groups and different Rhesus phenotypes (See Appendix I and 2)according to a prescribed protocol.

/

This document is not issued to the general public, andall rights are reserved by the World Health Organization(WHO). The document may not be reviewed, abstracted,quoted, reproduced or translated, in part or in whole,without the prior written permission of WHO. No partof this document may be stored in a retrieval system ortransmitted in any form or by any means -electronic,mechanical or other -without the prior written permissionof WHO.

Ce document n'est pas destine a etre distribue au grand publicet taus les droits V afferents sont reserves par l'Organisationmondiale de la Sante (OMS). line peut etre commente,resume, cite, reproduit ou traduit, partiellement ou en totalite,sans une autorisation prealable ecrite de I'OMS. Aucune partiene doit etre chargee dans un svsteme de recherche documen.taire ou diffusee sous quelque forme ou par quelque mavenque ce soit .electronique, mecanique, ou autre -sans uneautorisation prealable ecrite de I'OMS.

BSj90.1645Page 2

Per laboratory the potency tests of the proposed standard were performed with 4different cells in two different techniques: one prescribed method used by allparticipating laboratories, the other the commonly used method of each laboratory.Grading was done from 4+ to 0, scoring from 12 to 1: 4+ -12, 3+ -10, 2+ -8, + -5, (+) -3, +/- -1 (Appendix 3 and 4).

RESULTS

Uniformit~ of fillThe mean of the content of 24 ampoules was 42.1 mg. The standard deviation of

the content was 0.43. The coefficient of variation is therefore 1.0%.

The 95% confidence limits of the content of the ampoules is 41.2 x <43.0

Water content

The moisture content of the ampoules after freeze-drying was found to be 1.44%

Stabilit~

The accelerated degradation for testing the stability was done at threetemperature, 4, 23 and 37oC. The ampoules were stored at these temperatures for4, 8 and 12 weeks, respectively. When tested according tot he prescribed method forpotency testing against two different cells (0, R2 and O,R1R2) we did not finda difference in titre nor in score with the R2r cell and a drop in titre from 1:64to 1:32 with a drop in score from 50 to 45 with cell R1R2 only when stored at37°C for 8 and 12 weeks, respectively. The other time/temperature stored samplesdid not show a deviation of the reference values.

Sl2ecificitx

All the tests for specificity with 6 Rh(D)-positive cells of different ABOgroups and the Rh-phenotype were positive. The cells were collected from clottedblood and from blood collected with GPDA-l, EDTA and glucose citrate blood asanti-coagulant.

Each investigator also tested the proposed standard with all 6 Rh(D)-negativecells. All tests gave negative results, i.e. no agglutination was observed. Againthe cells were collected from clotted blood and from blood collected with CPDA-l,EDTA and glucose-citrate as anti-coagulant.

The proposed standard was tested by 8 out of the 9 collaborating laboratorieswith a rr-cell, sensitized with anti-c.

In all cases the test results were negative i.e no agglutination was observed

The samples which were used in the stability tests have also been tested forspecificity. All samples, irrespective of storage time and temperatu~e,gaye~~t =reactions with 10 roandomly chosen Rb(D)-poslt1ve cells of group 0, A and B. Allsamples gave negative results when tested with Rh(D)-negative cells of different ABOgroup and Rh-phenotype.

BSj90.1645Page 3

Potencx

The potency tests were performed with 4 different cells of predefined Rh-phenotype, all cells gave positive results.

In general the reaction with the proposed standard when used undiluted graded a4+. The titres ranged from 16 to 512 when tested according to the prescribed methodand from 4 to 256 when tested by the laboratory method (see tables 1 and 2).

The most commonly found titre was 128, both in the prescribed method and in theindividual laboratory methods (see Fig.l and 2).

In tables I and 2 the scores obtained per cell are also given. To get a betterimpression of the type of agglutinates obtained in the potency tests, thedistribution of the grading of the reactions, performed according to the prescribedmethod was determined (see fig.3).

DISCUSSION

The weight distribution of the ampoules shows a very small variation. With acoefficient of variation of 1.0% the contribution of this source of variation to thetotal variation will be negligible.

The residual moisture content of the freeze-dried ampoules is normalmoisture content contributes to the stability of the preparation.

A low

The stability of the preparation as found in the accelerated degradation studyis very good. The data found do not allow the determination of the shelf-life ofthe preparation when stored at different temperatures because storage for up to 12weeks at elevated temperatures did not significantly affect the potency andspecificity of the preparation. The small drop in titre and score as found with 1out of 2 cells when stored at 370C for 8 and 12 weeks, indicate that some loss ofactivity will occur, when stored for a long time outside a refrigerator. The longterm storage of the proposed standard is at -300C, as with all standards kept incustody by the Central Laboratory. The stability will allow shipment withoutcooling conditions, even if it takes days to reach the destination,.

There can be no doubt about the specificity of the proposed standard. Not asingle unexpected or an aspectific reaction has been noticed by the participants.Also tests performed with Rh(D) negative cells, sensitized with anti-c, gavenegative reactions.

The potency of the proposed standard is good. Some participants included acomparison between the proposed standard and routinely used chemical modified anti-Dcomplete blood-typing reagent. In all cases these reagents were less potent. Ascan be seen from the distribution of the grading, there is a sharp line betweenpositive and negative results, no trailing of weak reactions was observed.

As one of the participants remarked, this made her feel "uncomfortable withserial dilution titrations for the evaluation of chemically modified reagentsbecause the dividing line between strongly positive reactions and negative is verysharp; small errors in dilution accuracy may dramatically affect results".

The results of the potency tests in this study do not clearly reveal thiseffect, which may be due to the use of a clean pipette for each transfer.

Of the 36 titrations performed, 26 gave identical titres with the 3 duplicatesin 7 cases there was a difference of 1 titration step and in 3 cases there was a

BSj90.1645Page 4

A titre of 128 is the most observed one, even in the "laboratory method".From this one might be inclined to assign a potency of 128 IU/ampoule to theproposed standard. For a commercially available reagent a potency of at least32 IU/ml should be recommended.

CONCLUSION

Based on the results of this international collaborative study and with theapproval of all participants we recommend to establish the proposed standard as theWHO International Standard for anti-D (anti Rho) complete blood-typing serum(chemically modified) and to assign to it a potency of 128 IU per ampoule.

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APPENDIX 1

RECONSTITUTION OF THE LYOPHILIZED SERUM

Proposed anti-D standard. saline agglutinating

1 Cut off the upper half of the ampoule

Add 1-illl of distilled water and wait for 15 minutes2

3 Homogenate the dissolved serum by gentle mixing.

Although lyophilized serum normally does not contain a preservative, thispreparation contains 0.1% NaN3. Take care to prevent contamination, store at+4oC and, if possible, complete the tests within 48 hours afterreconstitution.

BS/90.1645Page -10

APPENDIX 2

SPECIFICITY TEST ANTI-D SALINE AGGLUTINATING

The specificity of the proposed standard for anti-D saline agglutinatingshall be tested according to the following protocol and according to thelaboratory method. This test shall be done with cells of the following ABa andRhesus groups:

BA, 0 Rlror (CcDee)

A B 0 Ror

0 Rlr

0 Rllr

(ccDee)or

A B (Ccddee)or

A B (ccddEe)or

Alrr (ccddee)

Brr (ccddee)

Orr (ccddee)

and with

A, B or 0 rr (ccddee) strongly sensitized with anti-c incomplete. Cellmust give 4+ reaction with anti-IgG and must be negative with AB serum inthe prescribed method.

1 Wash the cells one time with an excess of a physiological phosphatebuffered saline solution (PBS, pH 7.1-7.4).

Prepare a 3% suspension of these washed cells in PBS

Put one drop of the undiluted anti-D serum in a clean glass test tube(10 x 75 mm), add one drop of the 3% cell suspension, thoroughiy mix thecontents of the tube and incubate for 5 minutes at room temperature(20-250c).

4 Centrifuge the tubes for 60 seconds at a relative centrifugal force of200 g.

5: Examine macrQ §£°I! icall~cf5?;-agg1~E!:p~tc~on,

BS/90.1645Page 11

APPENDIX 3

PROTOCOL OF THE PRESCRIBED METHOD OF THE POTENCY TEST

Antiserum: anti-D saline agglutinating blood-t~t>ing serum

1 Wash fresh Ror (ccDee) cells and R2R2 (ccDDEE) cells at least twotimes with an excess of a physiological phosphate buffered saline soution(PBS, pH 7.1-7.4).

2 Prepare a 3% suspension of these washed cells in PSS

3 Prepare a twofold serial dilution series of the anti-D serum in PBS, towhich 10% of AB serum has been added (PBS-AB) (1:1 to 1:256). To avoidcarry-over, a clean pipette shall be used for each transfer.

Put 0.1 m1 of each serum dilution in a separate clean, glass test tube(10 x 75 mm), add 0.1 m1 of the 3% cell suspension, mix thoroughly thecontents of each tube and incubate for 5 minutes at room temperature(20-250c).

4

Centrifuge the tubes for 60 seconds at a relative centrifugal force of200 g.

5

6 Examine macroscopically for agglutination by gentle disloding thecell-button of each tube. The reactions shall be graded according to thegiven notation (page 4).

BSj90.1645Page -12

APPENDIX 4

POTENCY TEST ANTI-D SALINE AGGLUTINATING

Determine the titre of the proposed standard for anti-D salineagglutinating blood-typing serum according to the prescribed method andaccording to your own laboratory method.

The determination shall be done with 4 different cells. One of thesecells has to be a group 0 cell. Two cells have to be of the Rhesus type Ror(ccDee) and two of the Rhesus type R2R2 (ccDDEE).

Use the following notation for grading your reactions

4+ cell-button remains one clump

3+ cell-button dislodges into several clumps

cell-button dislodges into many small clumps of about equal size2+

1+ cell-button dislodges into finely granular but definitely small clumps;

negative

Doubtful reactions are deemed to be negative.The potency titre value is the reciprocal of the greatest serum dilution

for which the reaction is graded at 1+.

BS/90.1645Page 13

LIST OF PARTICIPANTS

Dr. Theis BackerBlood Bank Rigshospitalet9 BlegdamsvejDK-2100 CoDPcnhaQ@n

DENMARK

Professor S.R. HollanNational Institute of Haematologyand Blood TransfusionDarroczi ut 241113 BudaDe~tHUNGARY

Dr.

A. Chung, Head Serology Nat.Ref. Lab.The Canadiqn Red Cross Blood Society1800 Alta Vista DriveOttatJa. Ontario K 16 4J 5CANADA

Dr. P.Y. Ie PennecCentre National de Referencepour les Groupes Sanquins53 Boulevard DiderotF-75012 Paris CedexFRANCE

A. Mallory, MT (ASCP) SBBDirector, Laboratory ServicesBiomedical Research & DevelopmentThe Jerome H. Holland Laboratory15601 Crabbs Branch WayRockville. MD 208fi5U.S.A.

Prof. Dr. S. Seidl (executive dir)Blutspendedienst HessenSandhofstrasse 1D-6000 Frankfurt am Main 73GERMANY

Mr. D.S. FordCommonwealth Serum Laboratories45 Poplar RoadPARKVILLE. VictoriaAUSTRALIA. 3052

Mrs. P. Ann Hoppe, HFN-830Acting DirectorDivision of Blood and Blood Products8800 Rockville PikeBethesda. MD 20892U.S.A.

Dr. L. KornstadMedical DirectorNational Institute of Public HealthDept. of Immunology75 Geitmyrsveien0462 Oslo 4NORWAY