World Health Organization

fVHOfBS/O8,2i)93 ENGLISH OKLY

EXPERT GOhIMITTEE OK BIOLOGICAL STANDARDIZATIOS Geneva, 13 to 17 October 2008

Collaborative Study on a proposed WHO 1st. International Genetic Reference Panel for Haemophilia A, Int-ron-22 Inversion, Human gDNA,

NZBSG Code 081160

Elaine ~ r a ~ ' " ' , Ross ~awkinshand Paul ~etcall'e"

I ~iclt-h~rapetttics Group , and Cell Biology and fmaRitzR2. l%-atic~tzul Institute,for Biological Sturzdards und C o n h ~ l ,

Klutzche Inrize, South it;lirnms, Putiers Bar, f-lerts, Ii::W 3QG, LfK

Principal it~vesti~utur.'

O World Health Organization 2008

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\YHQIBS/O8.2093 Page 2

Summary 1;ourteen 1ahor:ttories ptirticipared in an international collahori~ti\~e study ti? asiess the suitrtbilit) of a panel crt'fortr senctmic-I3NA igIINA} ssmplei as the I h t 1ntemation;ll C;eneric Reference 13ariel fix I.f~lentophili;i 1%. Intron 22 In\.crsion, Fluman gDNA (NIBSC' code 1)81160). The panel corlsisrs ot'gDNA from a normal nlale 10611 861. a norm;ll knttlfe iOb!200 ,. a kmslc csrrier tO612t13) and an affecred male 10711 16). The participants etairtared the panel against their in- house controls which were previously characterised patient samples. I11 titttil, 165 genotype test., were c:lrricd OLII on the panel, with an error rate of 1.8 (Sr. The t-indi~lgs ut'this itudy indicated that this panel is suitable to he wed as a reference material for genotyping ttf I-iaemophilia inlron 211 inversion ntutation. The parlicipantx all agreed with the recomrtlencfatittn thrrt this panel of Ibur- gCfK1r\s should be pmposed to be est;thIished a.; the 1 "' Intentational Genetic Reference Panel for t laernophilia R. Intrctn 22 Inversion, IIuntan g1)NA.

Introduction flaemttphilia A is a hereditary ~ene t i c bleeding disorder and occi~rs in about I in 5000 to IOOOO nlafe birth\ if 1. The phenotypic manifestations are partial or complete abxence of circulating factor V111 (F-t'lil, F'k'j, a coapulaticin factor protein. leading tct frequent spontttneous bleeding into rhe jctints, muscles and intern~il organs. Correct molecular diagnosis, especially of the female carriers, is of paramount importance. In addition to transmitting the defective gene, rlpproxinsately 10% of the female carriers also have low circulating levels of FVIII and therefore are at risk of bleeding (2). lntron 22 inversion mutation of the F8 gene accounts Lbr 50% of severe haemophilia A, the most common X-linked congenital coagulation bleeding disorder (3, 3. 5). The inversion had been reported to be caused by an intra-chromosomal recitnlbination between a 9.6 kb sequence within the intron 22 of the F8 gene and one of the two almost identical copies located about 300 kb distal to the F8 gene at the telomoeric end of the X- chron~osome ( 3 ) . Generic analysis of the intron 22 inversion is challenging, involving technically demanding methods such as Southern blotting and long distance PC'K (6, 7). External quality assurance schemes have shown that errors in genotyping for this mutation do occur (8). Most laboratories use as their in-assay control DNA samples extracted from patients knotvn to carry the intron 22 intm-sion mutation. However, these are not well characterised and are usually only available in limited amounts. Stable certified reference materials are not available for validation of methods. We have therefore produced a panel of genomic-DNA (@DNA) lnaterials extracted from immortalised cell lines produced by Epstein-Barn virus (EBV) transformation of lymphocytes from blood s:tmples. The samples were obtained from two normal indit.iduals, an intron 23 inversion posititre female carrier and an intron 22 inversion positive male, with a view to establishing the DNA preparations as the l st International Genetic Reference Panel for I-laemophilia h, Intron 22 Inversion. Human gUKA.

The mrtln alm ot thrtl ~nternar~onal ccrtlaboratltr \tu& \\a\ tct ec;iluate this panel of 3 free~e- dned gDNA samples for their surtah~lttj a\ the U'I-I0 l \ t Internatronsl C;ertet~c Reference Panel for liaemophrtia A Intron 22 Inver\lon, tTuman gDNP,

This project Matl apprttced b j the Screntlt-tc and Srandardtiation Committee {SSCr ot the Internattonal Socler) on Thrombo\~\ and Siaemotlta\ls (IS ffT) rn July 2006 and ~ t l \ endorsed by the Expert Comm~ttee ctn Rlological Standardisat~on (ECBS) In Octokr 21X)6.

Candidate sampIes Blood \ample\ Rere collected lrclni lour con\ented donorb: each %a% tc\ted and found nepatrte tor anti-HI\, 112. HBsAg and anti-HCV. The genotypes of the donor\ \\ere confirmed h)

\VW OIBS/08,2093 Page 3

Srwthern bltrtrins rind Iong distance PCR. Epstein-Ban ainti was added to the blood samples to pritct~rcr irnmt?rttlliseci Iy~nphoblastoid cell lines. hl:ister cell stocks were accumulated and stored to ensure cctntintial ft~ture ,upplie\ itt' the same cells. C;eno~nic-DKA samples wa-e cxtracteci from cell peflers. using the €$entra Autiipuref,S I X A extraction robot t Qisgcn, C'r:.:1~ Iey, Sttssexl. The purir) of the extr~lctecl DXR tvas confirmed by cyrical density and ilgarose gels. The sa~nples Barre then kecze-dried in 5ealed glass ampoules. 'The citncentratio~~ of gf3KA in each anlpiluie \v;is estirnared by 011 to be approximately 7(10pgiml [ in IliinM T'risll.6rnM Et>?'i\iSClmgi~~~l, trehiilosel when reiitrspended in 3Opl of sterile wilter. The gen~3typr"s of the freelie-dr-ied s;trnples were funher 3n:tIysed and confirmed by Southerr1 blotting ;ind long distance PCR. The prodttet c11;lr;tc.reristirs are stta~marised in Table I .

Table 1. Product Summary

Code I >tale normat j Fernale tlormal j Femaic carrier i Affected male I

available l i I

n =l21 j Mean OD ratio I 1.89 i 1.89 1.86 l

Mean DNA conc.

Storaee Conditions 1 -20°C

custodian i This standard i s intended to he used in the irr vitr-tr diagnostics field and it relates to I35 EN IS0 175 l 1 :2(103 Section 5.5.

Participants fZifteen laboratofies agreed to participate and reiitrlrs were returned by 13 lahctratories fsitrn X different countries, Each iahoratoq has been assigned a r ide number avhich does not reflect the order of listins in Appendix I which shows the iist of participants.

Assay design Twelve coded samples of the panel ctf four gDXA materials were sent to ettch lahordtctq~. Each lahnraron; was asked to ~ r l i ) r m their routine genotyping test far the I-iaemctphilia A, intscjn 22 inversion and that the coded samples should be genittyped in groups of' four on three separate days as indicated on the results sheer, using dif'f'erenr reapents or different opemtors if possible. If

wH0IJ3S/013.2093 Page 4

the pa~tic~p~ltzf~g I,ibortiritr> ritt~tlnelj u\ed more than one genotyplng mefbcd. t t104~ method\, ciri~ld be c,irrred out in the \amc uiij on the 4ame \et\ ot ;i)lied \ample\

C)verall finding, of each sample and raw data e.g. phoro,nmphs of gels, amplificariitn plctts were to hc reriirned together with trill detail of techniques used. an!; in house or commercial co~ttrctis tincl reasons for failure of any of the \ampies genotyped.

Results

Testing carried out by participants I,,ibnraritr.ret were L~\ked to cdrf-y out the te\ting on 3 separ~ite da? \ and there uere 110

Itlb~r~itorrcrr uhrr cortln~cnted that the) Mere unable to do this. I'ert~ng utt\ carried out b) more than cme oper;ltor tn I Idh\ and 5 lab\ uere able to I I \ ~ more than crnc batch of reagents.

klethods used by the participants Thc methods usecf by the participants are listed in Table 2. All 1ahor;rtories used in-house meth<?ds: 1 1 labs employed lol-tg-distance PCR, 1 lab used inverse PCR. and 2 labs used Southern blottrng Thrrteen Idboriitone\ wed rn-house patient matenal or Dic-'tl. utth hnown genotype, a\ control\ and 1 lab d ~ d not ernploj an> control\.

Table 2. Techniques used by the participants

I Lab Rlethod 1 Method In Assay Control l

Lonf distance PCR I l intron 22 carrier female

l

j Long di\tance PCR j normal. intron 22 affected male, rntrc~n 1 - l 1 22 female carrier

carrier I ; 8 l 1,ong distance PC'R 1 2 j normal male, inrron 22 female 1 1 1 : DYA

% l

1 U Long di\tnnce PCR I 1 i\lrghr l lntrun 1 2 dftected male. intron Z? , modti~catrctn to 1 ternale cdmer, normal DXA

I l

I i I 1 3Xernale camer DNA l ! l3 1 i.onz dirrance PCK I l . ? I "iomdl male, lntron 22 affected male. I l 1 ~oudlerrt Rlott~np In-hcru\e 1 ~ntron 23 carrier 1

l / Long d~stance PCK and ' l 1 1 none

t 1 sep'irate gender

I l wing Abkotr f-raglie X

1VEIQIBSIOS.2093 Page S

%%ethod references I . 1, irt Q. R'ozari G, Sorniner SS. Single tube polymerase chain reaction ktr rttpid dittpnosis c?f

the inversion holspctt of mut~ition i r r Ilemophilia '4. Blood f 998: "3. 1458-i45<>

12. 1-iu Q, Sommer SS. Subcycling-PCR for mutripiex long-diitance amptificarion of regions uith high and low GC ctrntrnr: iippiictition to the inversion horspot in the factor V111 gene. Uioihecfrniques 1998: 25 1032 - 1038

3 , 1,iu Q, Nc?z:iri C;. Sommer SS. Errata (Primer mitdification). Bloitd 1Y98: 9.3: ? 141 4. Kossett I , t , Radic C'P, 1,arripa IB. de Braii C'D. Cienoryping the tlaeini>phjlia if~\rersion

hotspctt by uile csf inverse L3CK. C'Iitiieal Chen l i s t~ 1005: 5 l :7, 1 154- 1 158 5. Rctsslter JP. Young M. Kimherland Ml,. IIurter P, Ketterling RP. Gitschierx J. Eiorst

J,Morris hlA, Schaid DJ. de I\;lcxrlooue P. Sommrr SS, Kazaziitn, Jr.llli, Antorirtrrrkis SE £actor V111 gene inversions causing severe hemophiiia A or-iginate almo\t rxclusively in male germ cells. I-Iuman mvlecular Cienetics, 1994: -39. 1035- 1039

6. Pol5kov5 ti. Zmettikov5 I, Iiridasi L. Long distance PCR in detection of inversion mutation of F8C gene in Hemophilia A patients. Gen Physiof Biophys 2003: 22: 243-253

Correct results The 12 teit samples comprised four preparation\ and the following results Mere expected:

Table 3. Expected Results

Results returned by the participants r Thrrrecn fahi returned reiult\ ~denttcal to the erpttcted re%utrs as rndlcated in table 2

F One tab drd not obtain correct result\ for all three female csmer ianlples and reponed remlti, as nctrmal.

r Drtc to trme conktrarnr. one lab did not return rewits for sample4 1 t and 12

WHOIBS108.2093 Page 6

Comments from the participants Lab 2 - prefer liiluid IIKA, spccirnen reception st:tl'f wary of sla\x \ i d s . possibility for crror in tube tr;lnifer

I-:ih 4 - ~;iriation in I>K.A concentration. bt~t nor \ignil'ic;inrlj difkrent to thc normiil variation seen kztlvezn diagno4tic santpies on it ntn. I?til samples produced resr~its that they 3rc happy ro interpret

l-tib 6 - results iusgest cfiltrted DPiA s;tmples puni:tll! degraded in ;I couplc of ucehs ( 1 :5 ofthe 31fp l reconstituted volttme ). [;sing nlix I. unable t o amplify the 1 I kb friigment in samples 3. 7 and 1 I , In ;t second PC'K reaction with more LINA. faint band was rrccasionalfy ifkserved. FIIS this re;ison, an additional PC'K retctirtn lvith primers B and P were perforrtled li>r these samples, i\lso in thehe samples. with mix 11, the itltensity of the I t hfi AQ t*;tgn~ent wah syste~~~aticntly lctucr than of the 13 kh PQ frasment. These discrepancies were never observed in the carrier sa~nples analysed in our lab. .4pparently the lack or decreased amplification of some fragments in mix I was associated with increased non- s~c i f i c i ty of the PCR reaction (present as an increased smear in the gel). The order of the samples in boxes was quite confusing.

Lab 9 - results on agarose gel shu\\ed the qualttj and quantlt) of the DNA of the 2 sampIes kdrted, samples 1. 5, 8 appeared sI~ghtly degraded. samples 2, 1 1 and 12 contarned les5 DNA). nut iurpr~ringly. samples 2, l l and 12 gd\e ~ e a k s~gnals tolloutng PCR nin

1.A 1.3 - signctl with iamples S and 8 $+ere ueah in Sourhem blot and sctmples l . 2 and 6 shotaed \er> neak stgndl wrth Southem blot All iamples were OK urth long distance PCR

Discussion Fourteen laboratories. employing a total of 6 different methods with 3 different underlying principles. took part in this internationial collaborative study to evaluate the suitability of the proposed panel of gDN.4 samples as the I st Internatictnal Genetic Reference Panel fbr I I;~emophilia A Intron 22 Inversion, I-iuman gIl)Nh. The study was designed to determine how \&ell the panel may perform in a large number of laboratories. using a wide variety of methods. In order to assess the consistency of the panel's perfhrmance, the participants were also requested to carry out the study over a period of three days and using different operators and ditferent hatches of reagents when possible.

M rrh the exception (3.t one lahoratoq that returned ermneoui rewlts and one pdrtlclpant %ho d ~ d nor h,i\c trme to genotype all the ianlples, all orher piifitclpantit nlere ithle to genot)pe e w - 1 coded \ample correctl> Aif errors concerned the tnrron 23 tn\er%lon poitltlte fentdle ~ d r n e r I>\ 1 uhich %as genotyped d., a normal rndittduai W ~ t h 3 incomect result\ In I h6 testit. the cnerdlI error rate for t h ~ \ \tud> ~ d i l 8'~ The\e data ~ndrcdte that the mci j~~nr j ot l,tboratonei c m use thew mater~al., to ohtd~n the correct result and thdr the ptinel ,Ire sulrsble for trsc d4 reference ~ndtenals for norm:il female. normal male, ~ntron 32 in\erslnn pilstrrle female camer dnd lntron 22 incersion pos~tr-ve affected male C t ~ t h the exceptton of one Isbcirittof? that cird nor r-un known patrent \amplet as in-assd:, cnnrrol, ~ 1 1 other lahi hdle emplojed dppropnare as,;ly cctntrol, thereby ctrnllnntng the commutahtttzy of the panel

I-&h 6 ro~lnd the female carner sample\ rsamplst 4. 7 and I I t drd not ampltfy \%ell in their PCR reactions. nonetheiecs. correct results %ere reporled Thr5 I,ihorator~ aIso found degradatton

FlrHOIBSIO8.20Y3 Page 7

when rhr DYA sarnples ivere dittrted and left for a citt~ple of weeks. f:or tf-iiit rcasrjn. end-users will be ~dvi.;eii not to use stored dilutcd samples, L<:ih C ) frx~ncl slight degr;idatiorr \vith the

C affected n~ale DXA samples irriniples 1. 5, S), httt citrsccr resttiis iix there sai11p1es were obtained.

Stability study '1'0 eniLtrL. thc st;lhiiity of this panei, accelerated degradatictn st~idj i k hcing carried (rut on f'reeze- dried ampo~des stored at elevated tcmperaturc>. "1.0 date. agarose gel analysis of s;ir~iplrs that have been strtxd in etevated tcmperiitures of +-1S"C' and +Slt2C" fc3r l16 mirnths did no t show significar~t degradation whcn compared to sarrrples that hat e been stored at - 150°C. 'fhe stability of thc panel will bc continuitlfp monitored throughout the lifetime of these reference materiaiit.

?VHO1BS/f)lS,209 3 Page 8

Conclusions and recommendation 'Ihe results have shown that the panel of I.'i>rrr gDn',4 samples (a nortnril trlalc (OSilXli), a nitrrntkl kmale iOliI200). a female ritrrier iO6iZO-t) ancl a affected male (07iI 16)) eva1uatrd in this intematioi1;tl mufti-centre study is suitahlc ;n rekrence nlaterials for 1abor;rtories ctinying out gcnotyping for haemophilia h intron 22 inversion. It is therethre recommended t i t the par~icipanrs of the study and the experts on the Scientific and Sr;tnd;irdisation committee

tspccif~cally the FzVIIIIFIX subcommittee) of the lnternatiitnal Societ: on Thronrbosis and I4:temust;lsis (lS'fI41 that thii panel be put Ihrkvarci to the l3.pet-f C'clmn-rirtec fbr Bioii)gifal Standardisation ItC'BSI of thc World I-Iealth C1rg;tni~ation (WIIO) to be estabiishcd as the 1st

lr~ternittional Cienetic Rekrence Panel for 1I~iemophilia !\ Intron 22 Inversiitn. 1 I~irnan gDNA.

Participants and SSC experts comments to the collaborative study report 1411 pafiicipants agreed with the recommendation that the four gUNA samples rested in the cttllaboratite stud) should be proposed and established as the 1" LVHO Genetic Reference Panel for Haemophilia A Intron 22 Insession. tlurnan DNA. There was one additional comment:

* liselirl panel of standards, nicely reported piece of wclrk. Intron 1 will be particularly valuable as a foltotv-up given the scarcity of the mutation in the haemophilia A population.

All experts trom Sctentlfic and Standardt\atton comm~ttee (SSC) ttho responded to the report also agreed with the proposal. The SSC has endorsed the recommendation a the Annual Bustness Meettng (Sth SSC meetlnp rn Vtenna) on 5 full 2008.

Acknowledgements We would like to thank the following:

i Mike Makris (Royal Hallamshire Hospital. Sheffield. CK), E3ic Preston (UK NEQXS) and Steve Kitchen (Royal Wallamshire Hospital. Sheffield, UK) for obtaining consent from the donors and orgiinising the collection of blood samples

i The UK NEQAS fbr Blood citaguiation, Molecular Genetics Steering Committee for their advice Steve Keeney (Manchester Royal Infirmary, Manchester, UKf and Gillian Mellars (The Royal Free Hospital. London, l j K ) for help with prelirninar?; testing of the candidates

r The participants of the study > Mttlcolrn Hawkins for stability study and preliminary testing of the candidates

r W~llram Ptchering tor cell culture and bulk extraction ot pDKA r Ed Byrne ITDI, ?JIBS(') for EBV translormauon. cell cuItur-e and DNA extraction

References 1 . Soucie Jhl, Evatr B, Jackson D. Occurrence c3f hemvphilia in the t!nired State%. The

Ffemophilia Surveillance System Project Investigators. Am J Ifernatol. 1%"3&:59:288 -793

? P l u g 1. 2.!,1uier-Bun\chotL.n EP. Brocher-\.'nend\ Ail, \an Arn\tel E- i f i , \an der Bum JG. D~emen-tloman Jk. \.t'~llcm\e J. Rosendadt FX BIeedrng tn ciirnen of herntlphilld

UIocjd 2W6.108.52-56

Wti01BS/08.2093 Page 9

-3. 1-skich 11, Kazaci~rn Ill-I. Antonn~tkis SF;. Ciitchier J . In\tc~-sic?n disrupring the factor V111 gent. arc a cortlmitn cau4t. at' se\ crc hacmctphitia A. ".;at <&net. 1993:5:23(1-33 l

4, i"tnronar;ikis SE, Rttzsitcr JP, licttlng M et ai. Factor VIII gerx inversions in severe haemophilia A: result 01 3n internati~t~~al consoniur^ft 4tud:. Bloi~d. 1905::86:2206 -201 3

5. Kaufrr-ran KJ. Antorrarakib SE, 1-tty 13 ~2L?tf06) Factor V111 and henlophilia (4. In: C'ir1m;in RW et a1 iedii Iiemostasi.; ;~nd Thrombosis: B:isic 13rinciples and C"linic;tl Prricticc. bed L-ippincort-Rilven, Philndclpbia, pp I t ; 1-75

6. Rowen DJ and Kecney S (2003) Ilnlcashing the long-distance PCR for- detection ot'the inrrvn 32 in\'crcion of the factor V111 gene in severe hiiemofthili;~ A. Thromb f faernost. 200.1: 8931) 1-20?

'7 Rctswt Lf'. Radic CP. 1-arnpa IB. de Brabt CL3 Gencttyplng the hdernophil~d tnbenlon hot\pot by u\e ot Inrer\e PCR. CI~ntcal Chentrztr?. 2005. 5 1 7. l 153- 1 158

8. Perry DJ, Chodeve A. tIil1 M. Jennings 1, Kitchen S. Walker I; 1JK NEQAS for Blood C'itagulation. The IiK itjational External Quality Assessment Scheme (UK NEQAS) for n~olecufar genetic testing in haemophilia. Throlnh trlaernost. 2006: %:5:597-601,

CVHOIBS108.2093 Page 10

Appendix I List af Participants

11s S Keency. Manchester Royal Infirmar), h1:tnchcster. t!K t>rs %,l S Gnayar and B Tbcctphitus. Birminghizn~ C'krildrenr'i tftispital. t3irminghrrn1, l l l i Mr M Iia\sikin>. NIBSC', Potters Bar. l i K hrof Dr C' Mannhalter. C1inic;ti Insriture of Fi'frdical ;)nil C'hcmica! I>aboratoty lliagnostics, \Vien, Atrstria ISr It Saxena, 1\11 India Institrite itf hledicaj Siientei, New Delhi. India 1% 11 ,fterfini;l. Deplinment of lfaem;~ttrlo,ny, Royai Infirmary of [<dinburgh. Eldinbrtr~b, [!K 1)r A C;octdc.ve= Sheffield Molecular Genetics Services. Sheffield Children's NHS T r ~ t , Sheffield. l l K Dr I1 I-illicrap. Kingston General tfospital, Kingston, Onttirio, Canada Drs 91.1 Mitchell and J Cutler, St. 'I'homas' tIospitti1, l,ondon. [!K 13r 1% Sri~rtsta\~a. Christian Medical C'ollcge and i-lospital. Veliore, India Prof 1: Sa1v:ttore and G C:astoldo, CEI&C>E-Riotecnologie At~anzate, "Japles, Italy Dr C Vinciguerra. Hospices Civils de l,?;on, L-yon. France Drs L Gryesten Jensen and J Juncker, Ijniversity I-Iospital of Aarhus, Aarhus, Denmark Drs D 13)avid and I Morreria, lnstituto Nacional de Saude "Us Richardo Jorge". Lishctn, Poflugal

WH01BSlO8.2093 Page 11

Appendix If Proposed lnstructiuns for Vse

WHO li-tsrnabanarl 5tandnrd i s? in:enar.rrrd Ganc:ii Rriercncr Panei to- h&lemCjD,l.ii A

rnrrrrn 22 iluernion, Human gCPiA "r:BBC rode 08'iBC- ins t r~~t l l rns for 'isr

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9 REFERENCES l2 "

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l 6 IPIFORFrlATIOX FOR CUSTOMS U S E ONLY ...... --- ......... ... ............ Countiv of oriotn fur cuslomc- ourooaas' ' .;-'' k 8 ; .;-r

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Veterlilarf c~rtlfzcate G: ntner statement '1 opps;c imi. . "'~ac?=c_L... ...............

PJ;i::ana! Institute far Bielogieai Standards and Control