#~'K/~
~'l/
v
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Nordiskt referenslaboratorium
för genomisk blodgruppstypning finns i Lund
Av
AsA HIU.IDG.
AMmKA
HULT.MAmN L OUSOlf.
OCH EuSABI11I SJö.OG W!sTD, BLODCEJfT8ALE)f SItÅNIo UJlflVDSI1TJ'SSJUKHUSIT. L111'fD
L lnge har seroloai med hemagluti-
nation varit den alnande blod-
<2.-
grupperinpmelOdiJten. Nu lir Aven
'J genteknik tillgIngIia och i Lund finna det Nordiska referenslaboratoriet mr genomisk blodgruppstypnin..
I analyssortimenlet finna metoder mr att detektera de arvsanlaa som ger u~
hov tlU kliniskt viktlaa blodgrupper.
Laboratoriet lir det flnta i sitt slag som ack:reditera18 av SWEDAc.
Dryat 90 blodcentral...
Vid Sveriges dryJt 90 blodc:entraler ..
\
vinda hemauJutinadon mr att detekte- ra blodpuppsantipo och -antiJtropp8r.Detta sker i da& pi nlstan samma sitt som.pI tidigt 1900-taI dA Karl Landste- inert upptlekaren av ABO-systemet,
\ blandade blod fdn sia sjllv och sina
kolle... i provl& mr att se vad som skedde.
\ Mistan 30 blodgruppssystem
\ Blodgrupper bin av molekyler (protei-
\ ner, glykoproteiner, gJykoUpider) pi lerytrocytenl yta. Ett flertal av des.. ~
lekyler mretommcr ben pi andra cell-
l~I_.. Lex. ABO-IY8teme11 ltoJbydnt-
\truktuJer som finna pi mina- olib ly- er av Itroppena ceDer. F&' att en mo&.
yl ska klassificera som blodgrupp '~VI att den lrvI och mrtommer i ou-
~
varianter. samt att minst eD mInoisb lar bildat antikroppar mot ett antigen pi molekylen i MIL Utvecklingen fria Karl Landsteinen tid har lett tiU att idat r' ar 29 olika blodgruppssystem klnda.j Inom varje Iystem finna olika varianter.
Ii Se Fl"" . ABO-systemet lir det viktigaste ur1.
! kHniat synvinkel dl 8.k. natUrlip 8Dti- /' kropp8r (anti-A och anti-B) mrekom-
\ mu.
tiv, blivande mamma bildu antikropp8' mot sitt RhD positiva foster, som ..
ärvt sitt RhD-statuI frID pappIIL
Tvi seroloalska
analyser flire transfusion
Pro. 1: ABO och RhD-bestlnminl ut- mrs hAde pi blodaivue och patieut. PI givaren sker det i sambind med anmI- Ian inmr blodaivninl och upprep8I dir- efter vuje gin, en pile blod lInmu.
Prov pi patieateD ta LeK. i umb8nd med att haoIhoa sIt8I pi vllntelista inftlr opendoa.
Pro. 1: lDflIr tnnJfusioa
tu ett nytt prov pi patienten. En enkel bekrIfteIae av blodpupperinpa utRkl mr att k0n-T esterytrocyter
TID sIdUD8d fria teatreqenllIOID kap8 fria kommenieUa fabribnter. ertaII18 testerytrocyter tiD seroIoain fria speci- ellt utvaJda blodaivue. FI!r' att kunna detelctera blodaruppsandkroppw pi ett slItert sitt krlvl telterytrocyter fria blodaivue av blodJruPP O samt med vissa andpo i bomozyaot uJlPllttninl.
Hur urvalet ska ske
reaJeru
via ~ bot mr B1ock:entnJersom
pi ut av S venat Rk'eninamr
traII8fasiOlllllledi- cia.Andra viktiga system If t.ex. Rh.
Kell, Duffy och JCjdd. Blodgruppuys8e- men har ofta t1tt sina namn efter upp- tlckaren eller den patient dir uppt~
gjordes. V.uje individ har sin unika upp- sluninl av blodgruppsmolekyler och har IM gener mr de-- ay bId8 förIId..
V frekvens aven vi..
förekommer i olika populationer, tex. blodpupp A som fö- rekommer i 43~ hol vita, 27~ hos svar- ta, 28~ hOI uiater och i O'JJ hOI latin- amerikanska indianer. Med kInnedom om detta kan blodpupperinl anvllnd88 mr rlttsmediclnska och antropoloplb studier.
Idal If slvlU molekyl, len som i mAn.. fall funktion vllklnd mr flertalet blodpuppssystem och International ~
ciety of Blood Transfusion
har utarbet81 standardiserad numrerillJ och benIID- nio, av deua. Se Fi"" 2.Blod... p psan tå kro ppar bildas efter ImmuniserIni
AWtom ABQ.antikropparna kan ytter- ligare antikroppar bilda p.I.L transfu- sion, transplantation och/eller graviditet av den individ som exponeru mr en blodpuppsmolekyl som hanIhoa sjllv saknar. Antikropparna kan Je uppbov till hemolys vid ny transfusion eller . molytisk sjukdom hos nymdda (HDN).
Den mest vllkJnda orsaken till blod- Jr DIr en RbD nep.
trollen patienleD8 identitet. Dessutom utreda om patienten har bildat nAan blodJfUppsantikroppll sedan töregAea- de analys.
puppantibopp8r
#
c$
.,.,1
fenotypnlng med hemsggJulJnatlon och genotypnJng med PeR teknik.
Jfmf6reIH
meI8n
IARn8.Tn8.rr ,,/-.,.,u
004
RIa RH
K8I Duft'J KiM ~...
00t 001 18 0014
KEL PV lit DO
F1gw 2. EIlIHV8I bIodgtuppaystem, be_,."" de analyser som utmI8 vid /abc
, EndlJst V8llJ/glIiJtekomtnIInde.
GlylcolcOlljugat. lcoItydrall glylcolpld .,.Ier
Bng8&... ~
,
",
Utndnlnl av oldarhet.. Mir anvinds blodgrupp..
Om oklarheter
!OlDI.k. svala blod- genomisk typnln"
I"Ippavariuter eller antiltropp8r mot Vanlip indikationer Ir:
"
blodpu pper upptlcb. sker en utrednina 8 bestlmnina av fOltablodpupp (ro.
~
I .1mr att f&k.I... fyndet. Fram till mr c:a ter till immuniserad. pavid tvinna).
'-" 10 Ar sedaa IDvlnda enbart IICI'OIo8I 8 oberoende analys vid otl... raull8t med hemalllutinationsmetodlk. mea t.ex. vid ABO- eller Rh-pupperina.
numera fmna lvea moletyllrbiolo&i* 8 aJternativ till fenotypninl om p8Iieo- metodik att tilllA. Dock uvlnda oftut leD bir fia blod (lok. transful~
metoderna I kombination dl det lir en bllllJdbild) eDer lir poIidv med diRkt stor mrdelau tunna koppla resultat frta ag1utinationsteknik (DAT).
lenotypninl med aerol. 8 bekrlftelse av homozyaoti. ho8:
.
givare av testerytroc:ytllr.Blodcentralen Skin. I .
den bliv'" pepp8I1 mr aU bedömaAvd. för TransfusionsmedicIn rilken att fostret kommer att Irva --
Vid Lunda Univenitet finna A vdelniD- tipnet
som rnamm8IJIr immuniaerad
leD RIr tnnfusioasmedicia dir fonk- mot.ninl bedrift inom projebd
MdI1rIoW1-'.r ode skydtJsmdlllwlIWr vid bkHI- ProV'" flin hela vlrlden
,ruppsnltumliJ hlmoIy. - G~Mti.rktJ Vid nordiskt Overllkann&e 2001 utsJp och Junktio,,~l" stJUJi.,. Forsknlnp- den kliniska verksamheten tiD Nordiskt puppeD som ledI av docent M8rda L re(ereaalabcnlaium. men prov anu.
~
\ , Olaon bestlr (.D. av ytterligare Ana per- der Iven fria andra del. IV vlrldea.-' soner. en docent I medkinalt kemi. - Rutinmllsfat tal venprov I
rftrmed
"poIt-doc" ocb sex doktorander. V8l'8V EDTA-1i1l18tl. Ur detta kan man utvin- tvi likare ocb fyra biomedicinska Da erycrocyter ocb blodpuppuntikrop- Iybbr. GruJIIIeDI medlemmar Ir dal- par till seroloalsk analys, mea IveR utom verksamma Inom Blodcenb'alca DNA tiJllenetJsk anal)'L
SUne. VI,en menu forsknlnl och tU- AIr anaIya IV DNA kan aven an...
nik Ir dlnned mycket kort, I sjl1va verk. provmaterial anvlndu under flkutsIU..
et Ir mycket IV (onkDinpvertwnhetea ninl lat det innehll1er klmmrande c:eI- lokaliserad inom Blodc:entraJenl lat. ler. F'&' aU (utstIIII (osterblodpupp ler. Pill' aU kwlitetulkra den kliniskt kan provtapjnJ ske fria navelstrlnt.
diapOItista verksamheten i samma moderbb eller (ostervat18L moden som vid övrip Blodcentrllea En specien remi" Ir utformad mr lat sUne p.nsbdea denna IV SWEDAC slkentllla att aJl in(onnadon IV vik!
t6ten 2003. medföljer provmateriaJet. I denna an..
Flertalet metoder buer88 P' den ep bl.L typ IV provmateriaL pltientelll gruppena (onknina. I de (all andra ~ etniska dllh&iahet och tidfllre serolo- licende metoder anvlnda Mr de8Ia III»' Jiab resuII8L
delft UtprOV8ta, ofta som kandidat- el" Den kliniskt diaplOltfsb vertsamhe- magisterprojekt i samverkan med de ten skll8l IV biomedicinska anaJytiker biomedicinska analytilterutbildninlln18 frID BlodcentraJeaa laboratorium med vid Lunda Univenitet el1er Malmö beh&ipe.t att utll5ra och tolka anaJya-
HOpkola. Da. Efter en mrsta svm(onnuler!nl i.
lonystemet JI'InIbt deUa IV biomedi-
8
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TnDIpad 0kW
",., Nk bIodgtupp8genom ~ ~
cinst analytiker med hOI"' bel6ipea och slutiiIdII JOdkInnande
sker IV la.bre.
BlodgruppSI-nomi"
typnln. med PCR-teknlk
I mitten lY
1980-taJet publicendea Po-
typnlnl med Pel-teknlk
I mitten av 1980-taIet publicendea ...
Iymerue Chain Reactioa (PeR) IOID kom att revolutionera moletylllbiolo- gin. PCR Ir ca snabb. enkel ocb reladVt billil metod 10m fia snabb spridaiaa flin fanbUDI tiU kliniK dJ8pOIIit.
Inom flera vertsunhcfer 10m tliJUak ko.
mi, petit. miJaobiololi och patoIoai har anvllndandec av PCR-metoder mr- enklat och f&bIUnt befintUp
I11II.I,....
De... b8r mn...ttninpr ftIr aya
anaJY8IYPS' medlifta kInIIipet. Inom
tnDIfuaioaamedicia h8r utvecklinpa framm.. an. II1It PCR-buer8d HLA- typninl oc:b viroloailka anaIy* vid teIblini av blodaiV8nlo medan antalet blodpupplrelatende anvlndniDpom- ridea b8r varit reladvt fl.PI senare Ar har realdcU-PCR, eD myckd klaaUl mdOd, b&j. anvlDdll inom IranlfuJionsmecficinea. Med den- na kan friu fOlfer-DNA i dea pvida kvinnans pluma detebenI. Det inne- bAr aU den minimala halteD foster-DNA
ent181 oc:b
Deaa
blodpuppuelatende
ID'rAden h8r vuit rel8tivt fl.
PI leIW'8
Ar h8r rea)
na kall fritt fOlfa'-DNA i den pavida kviM8118 pluma detettera Det ma.
bir aU den minimala
balteD fOlter-DNA kan skilj. frID m8JlUD8l18. under RIrua- sAnnina aU de bida b8r aUb vlriau8erhalten
slnninl Itt de bida bir olika vllianfer av den undenöba ICDeIL Detektion av fritt fOlter-DNA i lDIJ1UIWJI piuma II' under utprGvninl i Lund eniiat den metod som anvlndl vid Intemadonella
vlrian8er
~
IDvlnda
ReferenaJabontoric (IBGRL) i BriIcoI U.K. Under 2004 kommer besdmnina av RHc ocb RHD 8U kunna etbjudaa som Idinist analys.
AnalyssortimentetiLund
Vid natinanalys IDvlDds nera principer mr lit dtenlllll resullllet
- nera potidoner inom vuje len uader- __L-
ReferenaJaboralari
bestImnint
...
.
LM NetmbeIp fr en av t»
bIom8cIIt:JtJ8a anaJyIIIt« som hM behiJtlglwla"
ocIIleUer
-
kombinadoDer av metoder si lC8PCR-ASP och PCR-RFLP utfln.
Prover som inte air att 10Ia inom k1i- nisk rutin
&Arofta vidare tiO lex. set.
venabeatlmninl mr aU finna orsaken tiD det avvikande Jaul...
.-
A Bo..y8t8m8t
Genomislt typa!nl av ABO ulfln RIt aU kunna raststIIIa padentens blodgrupp dl seroloaisk 1118lYI BeU oklara resuJ- taL Ofta rör eld Iii om mrvlrvllde eller lrftJip vlrianter med IV.,1re uuryct som benImn8 A- eller B-underpupper.
Dessa kan vara svira, dds&lande eller omOjlip aa definiera via serolopa.
Exempel pi fl!rvIrvade ABO-renocyper II' lvalt A-uttryck vid blodsjukdom el- ler Rlrvlrvlt B-andaea vid exempeMa sepsis dl bak1aieenzymer kan
omv..-.
la A-dien tiO B.
RtHy8t8m8I
RH-IYIferr'd bel. av tvi pner: RHD som avaOr RhD posidvlnepdv fenotyp och RHCE 80111 ler' upphov till and..
nen C. B. c: cd e. Den i vAr population vanliaUIe orsaken till RhD neaadv
~
utmI8 ~
fellOC)'p Ir toI8J avukn8d av RHD. I via-
. aadra ptIp.1Mw- f&åonuner ea inaktiv, "tyst'" RHD-leD SOlD kal...
RH D-pICUdolenea.
RH-typninl innebar flera anaJyser dir olib varianter (normalt. partieU. och svaat uaryck) av D- och CE-alleler iD- kl_w RHD-ptleudo bo deleltter&
Det Ir ba mOjli&l lit futsdlla om RHD-FfttlIJ finna i enkel eJIer dubbe.I uppllttninl (zYlosiaetsanaJYI). "'JOt IOID inte kan avlftru serologisb.
PAnENTFAU:
P""'.'"
FlIcka f&td -92 med ~I. 'fl I "*
rNijar utr8dd88 f&8t8 gMgen I ~
.
02.~Ir~ocft~
fund8t8d I hel T~
behcMllr ca en enhet ~ -_Ib" Y8' 14:.-
~. ~ ",.*" .-
fl , lida bIocfgruppun1Ic
av typ: anIhJk8, ~ 0Id8ra
,..
tIoner,
lr8nIfu8Ion8IIf med.".A. DAT...
av typ: i
donet. t
8f1&.A.DAT~
GenomIM .,....
ABa : M)t.
.RHCE: ~
FY: FYAM
JIC : .1(88 FY -8J8I8m8I
FY-typainl utflln vanligen pi pI'O\'II8- terlaI Ma (osta eller frID en 1r8nlfuD-
derId p8Iient men aven typninl
av 1aI- erytrocyIier ul1&l. Med typninaea k8 svaat uttryckande ella' "tysta" varianIIer av FY. som kan medflIra (abtt nepdva result8t i seroloain, detektenl. Deua ndnsbr risken mr att telterytrocyter (el-aktiJt Iolkas som homozYJOCer
avseeo- de andpnea Fy' och Fyb.ABO:
RHCIE : FY:
JIC:
~
Ale....
AB().gruppen luId8 l88Is1III8. Rh- och Fy-e/...~ Jar1d8'IIiIIII'"
och 8IdhJk8 V8rtII8r8d8L S8mnwI- IagllIdelIIIfter8d88 bbtgrupp och bIodgruppe8ntIc, vllk8t g8I'
_'.11IIan - bbt8nh8IIr.
KEL-ey8""'"
Typnina IV rOlla' tillllllU11lDl med bH-
vande
mamma och pIppIlr dea vlDli- gasteonWa tiU KEL-typnina.
LuoUTOIJIT )/~004
9
Antikroppar riktade mot KEL-aly- koproteinet kan av oklnda skll onab uualad blmnina av erytropoaen I k~
bination med hendy. vilket ler upphov till en slrsltllt fruktad fonn av HDN med utebliven retikulocytOl. Anti-K och Rh- antikropparna anti-D ocb anti-c II de som oftut ler upphov till kliniskt bety- delsefull immuniaerin..
JK. och .y8t8m8t
Analys av JK-aentn och genen somltyr uUyck av SI-andpnea (GYPB) pn i
ORDUSTA
AgglutinlII AIIeI8r
E rytrocyt8r ~ av entIuopp8r Olika varI8nt8r av 88mm8 gen
DlnIId agglutination 111 II
Del eam utIrycIc8 av 8IV88I1!8g8n 1ndYIdeI. 8N88I1I8g
Enkel uppeItInlng av en via ....
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etukdOm
ho8 nyIOdd8 Dubb8I uppdtInIng av vI88 ....Hum8n L8Ukocyt8 Anag8n PoIym8ru8 Ch8n R88dIan PeR med del Ipeclftk
pm.
PeR f61Jd av klyvning med rn8r1kt1onlenzym Kort. eynt8tI8k DNA-8trIng
<1('
DAT
Fenotyp
(r.t
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PCA PeR-ASP PeR-RFLP pm.
Lis M ElI
v
8d"Pw~
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akane ~BIodMnan
-
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FÖRFATT. 'ARMA:
10
mrsta hand pi transfunderade patienter dir fenotypen inte kan bestlmmas.
Inom J K kan Aven ovanlip "tysta" nri- IDter faststJUl...
r.u...1IIft
Kvinna f6dd
-
52. tidiga,. et tf8n8o.funder8d.
SeroIogI8II
typn...
ABO-fenotyp: AB, men med 8V8g8.
bI8ncIIIdIIb8nd re8kdoner med lU1II-&
~ SerumgruppIrtno
gen
V8r poeftIv mol B-188t8rytrOCy8Ir och negdv mol A-f88t8rytr0c:yt8.DO-8Y8t8m81
Realenl för seroloailt typninl av Dombrock Ir en bristvara. DIrmr kan lenotypninl vara ett alternativ vid ana- lya av slvll patientprov som IcStcrytro-
cyter. a
GInomI8IItypnInt
ABa : AlS
En nU8IIan
80m tIdIgIn 888008- nIt8 med ItIIV8g 8-Iubgruppd88IIt- t8f8dI8 I ~
R88uIIIt kunde r.e.. ~ "1 odt
~ lnfOr
tI'8n8fu8IaItrekommend8IIoIl8f
utflldl8.
P"".
Kvinna Mdd .73, grav barn 88dIn tIdIgII8 + SIraIcIgIIII..,...
...
K fenaW. K- . A.j"'~ ,.".
And
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r
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J/3004
Extended blood grouplng of blood donors with
,
automatable PCR- EUSA genotyplng
Maryse St-Louis, lask Perreault, and RMl Lemiewc
"",
1121 TRANSFUSION VoILme 43, AugU8t 2003
IMMUNOHEMATOLOGY
~ Hottcpff), r y
L ;~4
T be presente of the lA. B, and D bJood group antigens la routinely determined on RBCI of both blood donon and transfused patten.. to ensure compatibllJty.1 In blood transfusion cen- ters, testing la performed by antibody-based aglutination assa)'ll with automated equlpmenL However, RBC8 are not routineJy tested for all minor blood group antigena.
ThIs I1mJtation may resuJt In aUoimmunfzation of the patient against Borne of these antigena and will compUcate futwe transfusions 81 a resuJt of the need to lind compat- Ible blood. WIth the more frequent UH ofblood and agloB of the population, extended pbenotyplnt la more olten
required and 18 labor-intensive, since It il usua1Jy
per- formed with manual methods. Furthermore. it may be difftcult to perform on a iarge scale because of the non- avaJlabllity of suitabJe blood typinB reBBentl. For theN reasona, the procurement of compattble b1ood for alloIm- munized patien.. may represent a signiftcant operationaI problem If the patient Hrum containl antibodles apinat several minor biood group anti uIn the put 10 years, PCR auays have been developed for the detection of several gene polymorphisms respon- slble for human blood groupI such 81 Do C, C, E, and e (Rb):
K and Je (kli): Fy- and FY' (Duffy): and Jk8 and ~ OOdd).
The main Jmpetul for the development of thee8 8ISa)'II hU been the need to type fetal blood ce1l8 to evaluBte the ri8t of HDN.4-Iln the absence of RBC sampJes, PCR genotyp- ing can be conveniently performed on amnJocytes.l"lI
ABBREVIATIONS: PBS-c:ueID
.
l x PBS-O.25 pen:ent C81eJD.0.01 pen:ent tbbneroeII; PlS-T-I)( ~05 pen:ent~
From the Department of Raeud1 and DeveJopment. H....
Quibec; and the Department of Biocbemlatry and MkrobioJ.
ogy, F8cuIty of Sdenc:e8 and 1!nJIneertnc. Lawl Untversi~
SaIn.~ Quebec, CaDadL
Add1aI reprlnt requatJ tu: MaryIe St-Lo. PbD, ~P811e- ment de Rechen:he et I»veIoppement. Håna~bec, 253S, Boulevard Laurier, Salnte-~ ~ GIV 4M3, Canada;
e-maD: DIItIouidPhema-quebec.qc:.ca.
ReceMd for publk:ation September 30, 2002; revision received November 8, 2002, and lIttepted November 15, 2002.
TRANSFUSION 2009;43:1126-1132.
In blood ban1cs, NAT 18 now roudnely osed to detect contaminatlnl vfruse8. The detectJon of ampUcona olten reUea on fluoreacence technologfea. A le.. advanced, but more readUy avaUable, technJque Is the PeR-EUSA. in whlch dlsoxlsenin-Iabeled amplicona are hybrldlzed to.
spedfic blodn-labeled oUgonucleotide Immobillzed onta a microplate weD via streptavld1n.lJ..t TI1e presence 01 dlsoxlgenln-labeled amplicona can be deteaed with an and-dlsoxlgenln-conjupted reagent in a standard ElJSA.
simUar to the onea already in U8e in blood banb to det8ct vInl antibodleL
In this work. we have tested the poaslblUty of usln&
PCR-EIJSA to determine the Rh, Keu, Duffy. and Kldd blood llOuP antiSen8 of resuJar blood donors. "",1-" 11Ie reaults obtained show the suJtablUty of the method for extended typing of blood donon since only 0.3 pen:ent dlscrepency W8I obierved betweeD PCR.ElJSA-bued genotyp.. and antlbody-bued phenotypea of 100 bloocl donon for thelr D, C. C, E, e, Je. ko PyA. Jk', and ~ blood
llOuP and....
cC
MATERIALS
AND METHODS
Genomie DNA exlr8ct1on
A 1-mL pertpheral blood sample was collected from 100 randomJy 888lgned volunteen alter signatun! of an Infonned consent form approved
br
Hima-Qufbec'a Research Ethla CommJttee. Samples were anonymized.AlJquota of whole blood (200..,.L) were stored ovemlsht at -zo-c and processed for DNA extracdon with a DNA pwffication kit (QINunp. Qlagen, MJui888Up. Ontario, Canada), accordJna to the manuf8ctwer'8 speåftcationa.
Eacb blood sample wu phenotyped for all blood poup antlgel18 IIsted In Table l with standard IndJrect agglutl-
.
nation.
The RBCswere sensldzed for 15 minutes at 3rc
with commerdal blood pouplng reagents (Ortho-CIInlcal Dlagno8tk:8, Rochester. NY; Jmmucor. Norcrou, Go\; or Dominion BlologtcaIa. Dartmouth, Nova Scotla, Canada).Alter washIng. the pre8ence ofbound JgG was detected by addlng a commerdal and-human JgG reagent (Gamma Biologlc:aJa. Inc., Hou8ton. TIO.
(i
PCR 8mpllflcatlon
AU primen and captwe probea were syntheslzed by Ul8 Technologles (BurlJngtoD, Ontario, Canada) or SiBJD8- Genosya (Oa1cvfDe, Ontario. Canada). Sequences are listed In Table l. Esch blood group gene wu ampllfied sepa- rately alonl with s conttoI pne (four primen). The RhC and RIu: genes were. however. amplJfted. topther with . control gene u weU (sbl primen; Iee fil. l for detaDa). For PCR amplificadon. DNA poJymerue (AmplITaq Gold, Roche. Laval. Quebec. Canada) wu used aloOl with. l )(
PCR Buffet D (Roche). 50 11M dNTPs (Ufe Technologleal.
2.5 JIM dllOxI.mln-dUTP (Boehrlnp... Laval. Quebec.
PCR-EUSA FOR BLOODGROUP8
Canada). 1.5 to 2.5 mMMA (Rocbe). and apprmdmateJy SO ni of genomJc: DNA. The tbermocycler wu pro- grammed u followa: initial denatwadon at 94-<: for 9
minut-- ~d 32
cycles at 94-<: for 30 secondl, 48 to 65-<:for 30 seconda (temperature wrle8 between auaya). 8Dd 72-c for 30 seconda. foIiowed by a final eIonpdon step at
72-<: for 5 minut.. ~lndkated.
5 JII. of the PCR 1'88c-don wu loaded on a
..pen:ent NuSieve-apr088 pi to
verlfy the presence of ampUcona.Cetectlon of 8mpllcon8 by ELISA
WeJla of a flat-bottom mJcropJate (lmmulon 2, Dynatecb Laboratorfea, ChantflJy, VA) were coated ovemight It 48(:
with 250 ni of streptavidln in 100 mM carbonate
bufIw.
pH 9.6. WeUl were wuhed lix dmel with l x PBS-O.D5 pen:ent 1Ween (PBS- T) at room temperatun and blocbd with 200..1 of l )( PBS-0.25 pen:ent cuein-0.01 pen:ent thimerosa1 (PBS-asein) at 3rc lm l hoUl'. Mer bJoc:kiD&
5'-blotinylated specifk: ollgonudeodde8 were added at.
concentradon of 2°111 peI' weil in 100J&L
of PBS-C8IIIIn
(refer to Table 2 for the sequences ofbiotlnyJated olfsonu- cleotides). Some captwe oJJsonudeoddel contained a T- linker at the 5'-end of the lpeciftC sequence to obt8iD .hlgher
EUSA signal (bued on optimtzadon experfmenta performed in OU!' laboratory). The plate wu then incu- bated at 3rc for l hoUl' and wuhed six dmel with PBS- T.PCR product8 we", dUuted in PBS-cue1n and dJatrlbuted Into we. (equlvalent of 2.5 J&L
of ampUcona in
SOJ&L per weil) aloDl with 10J&L of IN N80H.Jb~
(o.SI!1&'mL. SIgma 0Iemk:81 Co.. St. LouJe, MO) W88 present ID the denaturlnaaI8P
u . pH IndJcator. After .
1G-mJnute IntUbation at room temp..tw8, tl!.e 0eutnJ- lzinl solution (3.0 J1!. ~f 0.5 MNaHJOJ wu added. The plate waalncubated for an addJdonaJ1 hour at eltber 3rc (El, D. and Kel]) or
ss-c
(C/c,~
and JCIdd 88&JI)CFI8- 1). We.
were wuhed Ipin
lix dmel with PBS- T.Anti-dlgoxlsenin-peroxidue conJupte (150 U ImIJ(Boeh- rfnser) waa added and incubated at 3rC for l bom In PIS- c:ueln. Att.
wu.hin&
~ the ortho-DhenvlenedJam1n8-- substrate wu ad_ded (Abbott Laboratorle8, MiasJuaup.Ontario, Canada) and the plate wu Incubated at room temperatwe In the dark for . maximum of 30 minuteI.
Reacdons were stopped with l N HzSO.. Mlcroplate8 W8I'8 read at 490 om, with a rererence at 630 nm on a plate reader (MRX, Dynex TechnoloBiea. ChandUy, VA). Reaultl were processed with computer software (Reve1adon 4.02.
Dynatec:b Laboratories).
RESULTS
Multiple. 8mpflflcdon 01 bIood group 8nd control geM8
Each DNA sample wu ampHfied in . serIe8 of 10 multi- plex reacti°~~ wi~ the p_rlmen llatecf In Table l. ReIer-
YoUIIe 43, A&1gu112003 TRANSFUSION 1127
ST~OUI8 ET AL.
enc:el
to pJ'8Yiowly pubUshed primer 8equencel or ...
sequences are IndJcated. AJ) PCR reactlons Included con- trola ~-actIn. human growtb hormone. or RHDlRHCB emn 4). AnaJysfa of ampUcon8 by gel electrophoresi8 revealed the presence of DNA fragments of the
~
8ize8 (dahl not shown). Genotypm, resulta Ior the 100 808-
Iyzed donan were compaMd to the phenotype8 obtalned by 8tandard serologlc technlqueL AnalysIa showed a 99.7 percent concordance, IndIcatIng thaI thia method can detect 8peclflc blood group genes with a high degree of confldence. The dlacrepanclea observed Invol\led thnIe
VcUn8 43, Augu8I 2003 1121 TRANSFUSION
donan with a false-poaitiYe lit
genotype. The methodwu round to be robuat
since only three PCR reactIona BlDon. the 1100 had to be repeated because of abenant result8. These three PCR reacdona wel'8 done on separate procedur-.Detectlon of PCR 8mpllcon8 by EUSA
Blotin.labeled captu18 oUlJOnudeoddes Immobl1lzed onto dfstlnct wells of streptavfdln-coated mfcroplatea are IIsted In Thble 2. The D and CI c 1LI88Y1 eacb used three dIt.
(,~ r MuJtiplex PCR
E e
~
@
Hybridization temperatures
3~C Flate l
PI.. l. Schematk: repraentadon of the PCft-ElJSA ..., deslp. DNA II atr8cted from wbole bIood, and a fnc:dcm II UIed far...
elfte PCR ampUftcatlon. Anneallnl tempentureI Bre lndlcated for etICb pnotype -,. A """t thermoc:JocIIr W8I
UIed to ...
lte twu plat81 anneaUJII tentpenturell'llDsJnI from 48.5 to 58.8"C for Plate l (E. e, Do Jr. and k) and from 81 to saec for""" 2 (JkA.~, Fy", fot. and C/e). respectfvely. For the PCR-EnSA detectlon. Pllte l hybridlzatlon II carr1ecI out lit 87"C, whle PIat8 2 II
hybridlzed at 55"C. Thll detdp - adopted to mab the ...,. amenable to automatloa and to reduce III8I1Ipulatloal.
i,
~ ferent capture probea, whlle the others used onJytwo (E/e, Je/le. Py-/Pt. JkA/JJtI'). 1Wo plates were prepared because of the requJrement for two different hybridJzation tempera-
tures
(37 and SSOC; Fig. 1). Alter a l-bom hybridJzation.plates were processed as In a standard
EUSAwith perm-
idase antidigoxfgenln conjugate and ortho-phenylenedJ- amlne substrate.Results presented In Table 3 correspond to the meaD OD obtalned for all antigen-positive donors as deter- mIned by standanI serology technfques. Negative con- trols,
correspondlng to PCR procedures carrled out in the absente of exogenous DNA. ylelded ODs varylng from 0.05 to 0.115. ODe of antigen-negative donors dJd not
PCR-EUSA FOR BLOOD GROUP8
200 ~L of wbole blood
~extrlction
200 ~L of genomie DNA
D K k C/c
exceed 0.280. except for JJtb. which save a mean 00 of 0.598 (range. 0.047 to 0.598). This high 00 il probably due
to a weak but nonspedftc ampllftcadon of the
JkA aDeJe.VoUne 43. Augu112003 TRANSFUSION 118
ST-LOUIS ET AL
.
to be quantltadve .. heterozysot8 and homozygote sampla showed slmDu OD signal&.
c!Z. DISCUSSION
Dur resulta show that an ElJSA performed In 96-.weU microplatea can be conYeDlently used to detect PeR prod- uCtl with hJsh bJood poup .pedfld~ ThJa concluaion 18 derlved from a comparatfve study In whJch . 99.7 percent concordance was observed between RBC phenotype8 and PCR-ElJSA genotyp.. ThI8 ftndJnl could lead to the Implementadon of an extended blood poup genotypq of bJood donon to racilltate the sc:reening of compatibJe bJood for aUolmmunlzed patient&.
The performance of the PCR-ElJSA was acceptable for roudne use. The DNA of andgen-posltIve donon yielded ODa that weie at leut 12 dm.. hlgher than the DNA of antigen-negadve donon for aU andgena except Jk'. The lower rado of 5.8 obtalned for Jk' was due to the relatlvely hlsh OD (0.598) ob8ened with Jk'-nepthe donon. The reason for thI8 high OD appean to be the presence of a small amount of JJtA-speclfic ampllcon gen-
1130 TRANSfUSION VoIume 43, Augu8I 2003
erated In IOme ~.nepdw IndIviduaJa that couJd be detected by electrophores18 after pI overIoad (data not shown). Since the Jk8/J~ poIymorphilm 18 due to a linsle nucleot1de mutation (G>A) at position 838, 18
thia nI8Ult
IUUe8ta 1Om8 weak nonapedftc primer extenlion.Desplte thIa background. 81124 ~.neptiYe donoR W8ftI acxurately typed by PCR-EIJSA.
An apparent faJse-poeItive reault W88 obtalned ror Py' genotypIng In three IndIviduaJa. We cannot naJe out the possfbUJty that these IndIviduals expreseecl a very low lever of Fy' antigen that wu miued In the eerolon te8tIna. 1b8
-- -
resuJta couJd aIao be due to the preaence of a silent Fy aDele In IOme IndlviduaJa."" Sequendng or genotypIns of the gene reguJatory elementa shouJd confirm thil poui- bWty. SInce the fa1ae..po8itMt resuIt wu obtained with only 3 pen:ent of the donon and couJd be euiRy detected by conftrmatory seroJostc phenotypiq. it il uncJear whether effona need to be punued to detec:t the liJeot aDele In the fint screenlns of donon by PCR-EUSA geDotyplq.
Bload group genotypel are usuaDy determlned by electrophoresil of the ampUconsln aprose pIL The main
advantage of the EUSA
format
Is the high throughputthat
can be achleved with automated equlpment. Indeed, automated equlpment for virul NAT and for. antlbody- andgen EUSA are aJready belng used In blood banka. The automated equlpment producdvlty could be further enhanced by using It for blood group genotyping. Since thil t.dog Is not requlred for bload quaJJftcadon, It couId be perfonned when the equlpment la not in u.. for the above teI88.Severm companJe8 are cummtly developlng auta- mated equlpment for mlcroarrays and for genotypIng.
However. thil equlpment il qulte expenslve at the present time. but 18 expected to become moJe affordabJe In a rew ye Meantlme. the PCR-EUSA deacrlbecl here could represent a Iultable alternative.
Jt should be point ed out that the PCR-EUSA format will provlde lesalnfonnadon than the currently used gel electrophoresla method. Indeed. for complex systems lUCh 81 D. gel electrophoresil enablea the direct detec:don of variants through the preleDC8 of ampllcona of una- peeted IIze or the absence of othe The EUSA can be adapted to deteet aD known variants
by uain&
muJtfple weUa. esch contaJnlng . varlant-speciflc oUgonuc1eotkle..However. the detecdon of rare blood group variants May not be neceaaary for large-acaJe genotyplng of bload donan, IJuce the RBC phenotype will be conftrmed before transfusion. Discrepandea at thia step could be. f\Jfther studied with gel electrophoreaia and DNA sequenc1n8 methoda.
SeveraJ operadonaJ iasu. need to be considered for the roudne ute of the PCR-EUSA method for extended bload grouplng. FInt. the donon will be genotyped onJy once. but thelr priorlty selecdon could be bued on l8Y8r8l crIteria. Second. an advantageouI characterisdc of thia technique Is the (act that the different steps (DNA prepa- radon, amplJftcadon. detectlon) can be perfonned at diJferent dmes. Msay throughput could be incre8led by accumulatlnl DNA samplee from seleeted donon before doinl PCR and
ELISA.
Another I.ue to be conaJdered II the need to dlatin-
&ulab
the phenotype and the genotype of donon In records. whether they are electronJc- or paper-bued. A phenotype Inferred from the donor genotype will onJy be conftrmed if the bJood la needed for a padent. Meandme.the Infonnadon would have to be stored In a separate box In the donor ftIe. Altemadvely, a prefix could be added to the andgen name to IndJcate that It representa a genotype (e.l-. afyA, gJt").
In conduIIan. the deveJoped PCR.EUSA genotypInI method represents a technolOl)' that could be routlnely used for extended blood poUPinl In the current blood bank environment wIthout major Investments In equlp- ment or trainlng. 1be eventual testins of aD regular blood donon will certainly facWtate the procurement of bload for alloimmunlzed patlenta.
tB
PCR-EUSA fOR 8LOOO GROUP8
ACKNOWlEDGMEHTI
We thanJt Claudlne C6t1 ror beIp ID coUec:dnt blood
...
AnNe See_jour for the serolOl1 work, SophJe LaPoInte for the oomparatM ana/yIt8 of gel eIec:trophore818 and EUSA. 8Dd 1e8D-
~018 LeIMoc ... rwIewtn8
the DWnIICIIpt.REFERENCES
An:e MA. ThomplOD ES.
w.p. So
et II. MoIec:uIarc:Ionini
of RhD cDNA
dertved from a ...
preIeIIt In RbD-po8dve, but not RbD-neptlve lndMduaIL BI~ 1993,-82:8I51-s.
Aubln Tt Le Van KIm C. Moum L et II. Spec:Ifidty and
....
sltMty ofRHD gmotyplnc metboda by PCR.1Jued DNA ampUflcadoa. ar
1
HumatoII997~.Avent HD. AntenaW
~
01 the bIoocI puupI al the fetua. VcmSant 1998;74(Suppl2):385-74.Maubnt-wnWJjIt PA. Ful BH. de RuJfter lA. et II. Geno- typInc of RHD by muldpla poIymer8e c:haIn ftI8Ctk8 anaIysII of aU RHD-1p8dftc ao... 1kan8Iu8Ion 1991;38:
1015-21.
Me)w
o.
HIJdebnndt M. SdIuIz B, et al. SlmuJt."MIOUI genotypIngol human platelet andpn8 (lIPA) 1 duo. 8 usIng new aequence-lpecIftc primen for HlM-s. 'DuItu- doa 1981;38:1256-8.Lee So z.mbu B. GneD BD, et al 0rpnJzad0n el the ....
enc:ocIIn8 the human lCeIl bJood group protein. BIood 1985;85: l... 7G.
0Iu0n MI.. Smythe Is. HIIMIOft C. et al 1118 P"f" pbenotype .. UIOdated wttb a mJ8IenIe mutadoa In die
Pf ....
predIc:tIJII AqtI8CJ8 In the
Dufr1 ~
BI' 11f8ema-tal 1998;10.'1:1184-81.
Lee So \Vu X. Retd M. et II. NoIecuIar buI8 of the Kel ~l) pbenotype. BIood 1995;85:912...
Rozman P. Dow: T. GuIner C. DUrlnndadon of autolapal ABO, RHD. RHCB, KBl., Jr. and FYbIood poup pIIOCypeI by anaIyII8 of pertphenI bIood umpJeI of padenta wb8 have recendy rec:eIved multiple lI'8DIfu8ion8. 1'ranafu8oa 2000;40:988-42.
Bennett PR. Le Van KIm C. CoI8 t et II. Prenatal ~ nation of feta! RbD type by DNA ampHftr-tIoD.. N
_I
Med
1993;329:807-10.Le Van KIm C. CoIta 't lIrOI88Id 't et'" Rh
~
dUeue of the neWborn and Rh lIftOtYpIng by RPI.P--ud aDele-lpedfic-PCR. 'n'an8fu8 CIn BIoJ 1995;2:317-24- Mu 1.( DI1Ien A. AIbano So et II. Intrlpllft aprelllon of
tranItormlna
powtb 1'8diIx- beta l by a novel quandadw reYeI'Ie transc:rlpdon poJymerue chaIn reactfon ELIS.\ In 1o..1udnc kIdney JedplentL ThmIpJantatlon 2000;70:812...
RapJer IM. VIIIaman.o Vo Scbochetmaa Go et II. NoanI- dioac:tlve. c:oIorlmeatc microplate hybrtdJzadon IIIIIIJ lur detec:ttna ampUfted human bnmunodddenc:y vIrUI DNA.
am 0Jem 199!1;».244-7.
l.
z.
s.
4.
5.
8.
7.
I.
8.
lo.
11.
12.
13.
VoIune 43, Augu8t 2003 TRANSfUSION 11 S1
Fletcher HA. Barton Re. Verwelj PI!, et al. Detecdon of AqwrfUlUl fumfptw PCR procIucu by . mJaodtre pl88e bued DNA hybddJladon -y. J C1n PathoI1998;51:
Paa BH, SImIek S. Bleebr PM, et al. Rh E/e genotypInI by aDeJe.lpedfIc pr1mer amplUkatlon. BIood 1995;85:829-32- Maultant-vanWljlt IlA, Fua BH, RulJter JA. et al. Genocyp- ini of RHD multipla poIymerue main reectIon
anaIJIIII
of III RHl).apedftc aD'" 1\'anafusIon 1998;3ltl015-21.
SInJIeton ax, Green CA. Avent ND, et al. The prnence of an RHD peeudocene containlnl 8 37 bale pair dupllcadon and 8 nonaeDM mutadoD ID AfrlcanI with the Rh D- nepdw blood JP'OUP pbenotype. Blood 2000;95:12-8.
17.
~I
1132 TRANSfUSION VoIume 43, AuguIt 2003
11.
19.
OlMs B, Mentman Mo BaJJly P. et al The molec:ular buk al the JCIdd blood poup poJymorpblam and lta 1act oIlIIDd- adon with type 1 dl8betel1UlCepdb1Uty. Hum Mol GenIt 1997;6:1017-20.
0ItI0n MI.. HlDIIOn C, AYeIIt ND, et Il A cl1nIc:aIJy appIc- ablt me1hod Ior
detaminID8
the three major a1IeIe8 8t the Dutry (F)') blood poup locu8UIIn8
poIymenIe cbam 188:- don with aDe1e-spedfic primen. nmafuIIon 1998;38:U18- 73.
'Ibumam11Ie C, Le Van KIm C, Gane P. et al AlJ89CYlIUb- stltudon resuJlIln very low membrane apJelllon of die Dutry andprecepcor for ~ In fy" lndtvIdulll.
BIood 1998;92:2147-56. EJ 20.
're
~
c
~"'
. .'"
c
(C
,_o
~
(
Cc
le
"'°.
fil'0,0
~
~
~
c
(C'
t.
«C.
'"
~
IMMUNO BIMA TO LO GY
I Genotypfng of RHD by multipla: polymerase
ehaln reaedan anaJysls of six RHD-specific exaDS
.
P.A. MQ/lSkant-van WlJl; B.H.w.Paa.r,].A.M. dsRuIJtBr,
AB. G..lT. von dem BorM, DJ, van RheMn, cwl CB. INUI d8r Schoot
ndIpIa
rectId
ml{,tlt
et
0v8rbeså, M.AM.
"
1 1M!U1 DJOoa group syBt8ZD II or CUDJcalImpor-tII1C8 becau18 1U1~ lpfnIt Rh lJIt8m an-
.
dpD8 are Jmolved ID hemo1ydc traD8fu8IoD
18- actioD8, hemolydc dI8eue of the D8Wbom, mi autoJmmunl hemoJytJc anemJL '!be Rh syatIm andpDI 11'1 expreaed OD po1ypeptldel eocodedby two hi8blY ho.
molOIOUlleDeI,
RHD md
R.HCIr.whIcb
111'8locaI8d on
chromoeoml1pSU
- 98.1.1 RHCBgtveI
die to tbe CIc 8Dd BI e polymorphllma, whDe RHD eacod.e81be D polypepdde.RAIca1tIJ
Jt hal beea abowD tbat Clc 8Dd BIlantfptw 818
~.Id on the 88ID8 proC8ID. wbk:b IDdlcat118 thatRHCB producee one produc:t' and thulmfut118 the hypothe118 cbat C/c and Ele andgeD8 uiIe by altemad.,. Iplfdq oftbe RHCB product. 11btal or pard8I deIetkm. of the RHD ....
C&D mru1t ID the D- pheDotJp&'" R8ceDdJ & point muta-
don paemttna an fD-1rIme Itop codoa In ... 1 of BliD
bal been reportecI to C8UI8 tbe D- pheDotype.. ldor8o98r.elpecfallyln
blackAfr1caD8and JapmeeelndlYlduall, ft8O
lI/11h)
ABBRIVIAT1ONI: ASPA . d8-8pedk pdmer ...,pllllc-doa;
CLI.
LaIJoratolJ 1br ~-.J lDd CIDIc:8IlmmUDCIiaV.Depatmmt of ~1m8maI1mmuDoh8ma1DlalJ.
SaaufD
BIood SappJJ PouDIf-"""'. .A~I" MedbI Ceof8r,UDivuIkJ
of Aaurardam: lSBT. Jm8madaDI1 Sod8ty lir lIIoocI TrIDIfu.
doa: MoAb(IJ .
monocIonaI~
NCR. ""~
.. - --'---
J8IIOI1i -
ft;K- .
--~--~
c:II8ID--_o
IWQIUII; ~~ D1IIY8'I- .
--' 1811 -'II.'CIaW.Prom dJ8 BJood B8nt ~ lDd tb8 I..8bocdDIJ lir BIp8d- m8Dt8l1Dd 0IabI
r ~
J)epIrtm8Dt of~
ImDND""em.ankv.
SanpID BIood SUpplr Potl~ Al:»- demIc MedIcal C8Dter,UDIMIkJ
al Amlcerd8m (UJ. lDd cbe Dep8ltID8Dt 01 HIIDåIIDIr. AademIc MedbIc..,
AmIt8rd8m. the N~ lIId the DIputm8IIt ofHemaad- og, UD1wnJty HospbI JIDa8rdIm. Ro lb8 N8dJa0-
...
'Supported ID plit
bJ
Glam DUm- 28-2S11 from 1'b8 Netbm:laDdl Pcnqvt.fIon far PnftDdv8 MedJda&Jt8caInd
b puhlcadaD 11DU81J 21, 1911; mIIIcm l'8C8Md May 15, l8, 11X18a:8pt8d..., rt. 1-'DWISPU8ION
1998;58:1015-10Z1.V0bn8 se. /IIoYemb8r1D8C81998 TRANSFUSION 1018
~
MAASKANT.YAN WlJK lY AL.
intact RIm senel can be pl'888Dt ID 18 non. ".U
Becau8e of ItlltrODI JmmunopnJdtJ D 18 tbe moct Importmt u;1dpn aftbe po1ymorph1c Rh iydem. D sbowI quantftlttve md quaI1tat198 variatfoDL Qumtltattn vufa- dona are c:haIuterlzed by eitber a larp Dumb. of D anti- gen attel on the red ceU (BBC) membraDe,
u
In D-""\or a amaJl number ofnormalD andpnsitea, u InweakD.U.lIlt bu been sugllt8d that quaJfta1fv8 D varlantlere cawed by two gmetic m~.nlam.. pne-convenIon evmtl be-
.
~~f!'
dpla4tl.
In tht8 report. we pneent. multlplu RHD typin& ..
..y bued on . rn plfftt-.uton of the RHD-aped1Ic 8IDD8 S. 4-
S, e. 7, and 9 tbat
WII pertbm1ed ID ODe reacdon mhttml RHD elDDIl, 2, and 8 and tbe c:odfnl reslOD of eJDD 10 111'1equ8l to the COIr8lpOncUnlRHCB aona, and thelr ampJ1I- cadOD la therefore Dat specl4c for RHD. Becauae al emaI with RJID. spedfk: 8eqUlDCllm tbe codtna regIoDl are In- volved In thJa PCR tecbntque, the cbance that doDOrl cu- ryml partlll D antfpDIlIW fa1seIy typed u havIns
a oor-
mal D 18 peetly reduced. ID addltkm. aDeIe-8pec:IBcpm.
ampUtJcatlon (ASPAJ WII developed forpnotypmlofD-, wbich la characterlzed by the T329C mutatloD In e:mn 2 of
RHD that resulu ID a leuclne-to-proUne subat1tu11oD at amJno Idd poåtIOD 110..
1 018 TRAN8FUBICIN VoIum8 38, Novemb8r/December 1988
MATERIALS AND
IVI" Il8nlA~ ""nu 'W'Ioo """,",V
Samp188
mca of119 dODOIlwer8 Rh pbenotyped acc:ord1ntto ltD- Wd protocoJa with moDOcIona1antibody (JdoAb) MB-201
recosntztnl
D epttope 8/7 (DepartmeDt of J!Ipertmmal rmmunohematolooSIDsutn Blood Supp)y Poundadoa
[a.B), Amsterdam, the NetMrI.~ and a polJclona1l1Jd.leIUID (antl-D bromeUD. CUl).
PheDotypJng for partlll D
ni pedormed by1lIin8 a panel of ee1ected MoAbI wIIb mown
ep1tope rpecUlcltJ D varlmtceI1I wen di8trlbutId
~ the 'IbIId Intematicma1 Våbhop
on MozJoc:Icm8I AD-tibodl. aplDat Red elD and Related AntIpDI CNam..
Prance, September 1998) or from the stock of frozm BBQ at the CD.
Of the 119 dooora, 53 \ftI8 wh1te, 48 were
~
2 [Donan ISBI' (International Society ofBlood 'Jl'Im-"~Lf and ISBT 80 from the NIDteI Wbrbhop) wen of UD-
mown
etbnJcbacqroUDd,
and 18 Mm 8elect8d becaI8th8f
WIII8 known to haw a D ftrlmt. Of the 53 wh1t8 elD-""..
~-- n ,,-..I...U '1"\ - o-, -.
suoJosScaDy D- do-
and W8rI
"
',:
D lmmuafadon muldpluRHDt]
8 RHD-specI1Ic eJ
'Ibe
Multfpla PCR IftIIly8Ie
DNA wu l8oIated
from perlphenl b1ood wb1t8ceIII bJP-"
nol-cb1orotorm-laoamy! akohoJ emacttOD accotdfDI
CDstandard methoda. SIx RRl).spec:I1Ic: primer Må WIll
dl:-
..
8Ipd to amp~J~tRHD~D8 ~ 4, 5, 6, 7, and 9 ~ 2). Al
prlmm wert RHD spedDC at theJ: 3" end. In six primm. Jr proved to be necesaaryto Introduce a mfsmatch near the
r end to .vald ampMcat1on of. product from RHCB. Se- queDC88 and poaldoDl oltbe primen are Hated ID 'IabI.t L.
a. a .
t .
Exaa
RHD
"..
R
""
".
I
"'" . DM8 .
".. - D8T - Ro- -
=-- Daller
1077 . IJ-pUn0tJpe8 bowD 8t pr88t muldpJa PCR technIq1I8.
bJ
black box8I. 0p8a ~ ,doa mark (I) JndIcat88 dud partIaIblack
npnnat8d bJ
(I ';fIa
-
-..
11t bp 128 bp 117 bp 17 bp 18 bp
RHO MULTIPlE)( PCR ANAL Y88
P8r muldpla PCR, l PI of pnomlc DNA WU Uled ID SO pL ofll8CtfOD mil: compoaed of 0.2 mmoI per L of e8Cb dNTP (Promep. Mad!8on, WD and 2.0 U of 7bq DNA paa,.
merue
CPromepJ In the approprtaq butf8r aupplemaat- ed with U mmol per L of MA; 100 DI of primen R848, RaSAD2, Rex9SD2, and R1219W; 25 DI of prbnen R38f.R474M. R97S, RIO6&, lUI98M, and Ral8AD3ö 15118 ofprlm- flrllU96 andR621j and lODlofprlmen~ aodBACIM.
Primen BACn CfoIW8Id pdm8r: 5'-ccrra::rooGc:A GAGTa:1'G-3') and BACTaa (moeqe pdmer: 5' -GGAGCAAr,.
GATC'lTOO'CTI'CS'), pueratfDc a200-bp product of the,.
acdn pne, ~ fncJuded u 8D int8mal CODtrol to noId fa1se..nepd'Ve reeuJ1& Bebe uch multtpJuPCR. :DJq DNA po1ymerue wu fncubated with a DeutraJfJ:fq MoAb to ., DNA poIymerue (nqSt8rt Antlb~ Cont8eh LaboIa.
rle8, PaIo A11D. CA) for a bot-8f8rt PCI. .
'Ibe multipla PCR \VU pedbrmed in . tbmnaI C)'å8r (Modal", Peddn Blmer C8tu8, Norwalt, CI') with the fbi..
Jowm, CJcle spedfk:atf0Dl: dmatunt1on tur 5 mfnutel at 9~ 52 CJde. of l nrlnut8 at wc. l mJnute at ss-c. aad 45 .ec0nd8 lit 72"'Q and ODe CJCIe ol5 mfnut8e at 72-c to comp1ete ateDI1on. PCR pmdudI W8n 8fz8-eeparat8d br electrophoreaflln a12-percent acryIam1de pi and vf8uaJ- Jzed by ethIdIwn luom1de ltainbJI.
Sel ,
'Ib detarmJne the ell'8ct 01
low8rlnt
the amOUDt of ampWlabJe DNA for the multlplu PCB. 2 pL 0I88rfa11-1D- 10 DNA diludonlransfDI
from 500 DI perfIL
too.oas
DI per pL from a D+ lDdJvkIuaI (DccBe) WII U88d u 1Dput.Ow ASl'A
An aDeJe.apecl& pdmer set wu tt-'sned to be co1DpO8ld of prfmar R258 (forwud primer:. 5'-cTI'GGTGTG- CAG'I'GGGCAAT(X3') and pdmer RDYII (revene prfm8r:
5'-c:c8CC1trcc"tacC'l'GTAm-S') poeratIns a 9S-bp rr.- ment. 'IbIS' lind Ducleodde ofRDVII wu spedftc for DU- cJeodde S29 of the D'" A mjpn8td1 wu 1ntroduc:ed atDuc1eodde 332 to fnCRll888peå&ft)t 1b avofd fal8e.ner- attw resul1I. 8D interna! controJ ampJlfylng CODIenstJI fllDD
l (187 bp) wu 1Dduded.1be IalD8I8- action mfxture u d88crfbed for th.
I 10 muldpJaPCR wu UNd with the ad- dfdoa 01100 DI otpdmen B2581Dd
w-o- RDVJI and intema1 control prlmaa.
. -
Cyde ~ODl W8r8 15 mfnutll71. at 95-c. to1lOW8d
br
30 cyde8 of 45 sec0ud8 lit SS-<:; 4!1 secondI at 8O"C;I renne prb- and 45 l8CODd8 at 7rC. .BztenaIoD n ~I~ wu complet8d c1urtq S mlDuteI at r. VerdC8J 72"C. 7bq DNA poJymerue (Prnm..
I
UDfDo ...
II) WI8 8ddeddurlnstbe
ftnt 15 mm- JUlD.IDrffIa utellt src.. . il
,
~
.. -
Volume 38, Nov~ 1988 TRANSFUSION 1017
~
MAASKANT.YAH WlJK ET AL.
. I Z J . . , 7 . , 18 1112 U 1411
200 bp 100 bp
fil. So Mul II'" PCJt"""" of putW D ".. C8UIed ." tGUI- putI8I RHD
- _o - -
..,
DY- IUfUI"""" une J, DNA Z. D+r I.- ..
~ ... DF7tIlaM 5, D*J laDe .. I1"
Iype fr lADe 7,'" r,pe Ila Lule 8, D""II.-.. om-; Lane lo. Dl1'I1ane J I, .0 ~ L8ae 12. Do... SPI"'" 13, ~ 1077; LIIae 14, ~ L8n8 1 S. _t- controL IC. lD&em8I
CODtnIIrn. die
!to ecda...
. 2GO-IItI1'npI8nL
~
~;
.".'J. .'"-, .
RESULTS lIuldplex PCR anaty...
Frmn aH DNA samples. the 200-bp In.
temal mnrfal fragmenr from the ~
EpIDpe' actln gem ".. ampllfted. From DNA lIID1of aU 40 whlre D+ Individuala and ..
&om 44 of the 48 nonwhlr. D+ IndI. :::
vtduaJs, RHD eDna3. 4. S. 6.7. and. ...
were lenerared (Al- 3: Lane 2). ,.
nonwbJte donors ~re. aCCordiT1lID lpOIW7 standard protocols. serologicaU, IIIDI phenotyped - 0+. \Vhereu muJd- epOI pin KR DNA anaJysfl unexpectedly .
MoAbISCshowed the absence of RHD uon SID :::w s::
Donor 1579, wh Ich indlcated the D'* s.r.. Be genotype. and the absence 01 RHD BI
emRI 3 and 7 ID Donor 981. which la
=
In apmnent with the lY'" genotypl t """'"
(FJs. 3: l.anea ~ and 9. rtSpect1veJy). ~ . 1018 -m AHSFUSION VoIume 38. ~ 1-
1(
The predkted aenotypes W1ft CG8- ftrmed
RCOIop:auy
with die U8I.,
MoAbe ..
deecrlbed In ~bIe Z. ID.de«I, Donor 1579 hu the D- pbeDD- type and Donor 981 the [JI1III p---- type.
None 01 the RHD exoI18 WI!I8 ampWled from DNA 0113 whlre D- Individua18 (lncludlnl Donor .813-
Rh",.). All RHD-spedftc no..
WIft ampHfted from the DNA 01 dCMWf LSJIT J'" u expeccedIn .
weU D d0-nor. RHD exon 9 was ImpJlfted ironi the DNA of Donor ISBT 60 (C"ceeJ.
From DNA al all D wrianllleleed. me
pmence or .~ of RHD.8p8dIc
exons wu u rxpected from cbe pro-
pmed
pnotypeI (Flp. I 8nd 3)..IC._J ._4
'- J
--, .-
--,
Sln8l11vll'r
The mJnimaJ DNA Input for thlt mllhlpJel PCR lechnlque WI5 round
ro be 10 "g. whirh Is eqWvalenl to about 1000
cåIa. A DNA Input leif .han 10 "I resuhed In the !ou at sna8J PCR ~t.-d\.1fs. nona
3 and 7-wherasIM
kltanaI con lrOI frqment wa stiD Imp""ed (fis. 41.D" AM' A
From
DNA ofrour DYl' donan. alJRHD-sped8coonswere
ampfifled wltb me muilipIe. PCR (rmJlu not shown). DNA,f !bese donors wu aJso anaJyzed In the OJ" ASPA. From aD blrdonon. aD"'-5pedfk produc:t W8Sgenerated.
whm8s
oaly Ihe 'nterml coniroi rnpwnt W8J obtalned In nomW D+ md normal D- donoI'lIF18-5).I
.
DISCUSSION
ID lh8 repan. we pRSent .rast and rftIable ~thod ror RHD
~
The multipla PCR &edm~ Is bued"oo die ampU8cadon of RHD exona3. 4.5. S. 7. and 9 with the a.-vi RHD ~-spec:Jftc
prlmell. PrImen 1ftf'edesi8ned
la lUCh . way dIat aD prnentJy known genorypea 01 qual- lItIw RHDvulants caused by rot&! Of panJaI repIacement ofRHDaoo3. 4.5.8. 7. and/or9bythetrRHCEequtYaIenD Is IeCoptzed by me presence or absenat of their ampUI- QIIon produca.
Nudeodde cti1re1et1Cel between RHD lOd 1IHCE
have
been U8ed 1ft RHD typIl1I -ya. BenDeter aJ.
11 ueed le-cruence dUferences In the 3' NCI 01 RHD and RHCB. Nc8 et 11.18 deveIoped an RHD typinI UA)' bued on dw f8ct diat RHCE 'ntron 4 la 18."181 man RHD inoon 4.. Sequenc8 dllrerencea in exon 4 ud 7 h.w a1ao been used.I~14 tf bu
"--- u" .. - "- -
-I ~ eac.btlshed thac the use o'on~ lpedIt primer set for
~...:
L Dtyplnl may pnerele faJse-neplM and faJse-poådw
'"_""""31 Same D- donoll (~r' or r'r) will be typed RHD-potltJw in the 3' NCR ...~ uir ha, been abown lINd bybrtd D-Ci-C? whOl8 Q &eqUence spana esons 2
. II 200. bp100 bp
12345611
""
Determln8da1101"""" DM"'" lir ~ ra.
I, DNA naubn l.aH8 2
..
1, DNA lIIput 011000. 1 DI, II,.. 0.1. 8nd 0.0 I 118 rep-.d ,. LM8 I, COIICrDI. IC. ..
COIatrollraa die J-ac:d8 ..
paenI'".
200-" h8---.
RHD MUL TIJILEX PeR ANALYSII
Iresent in lhose donora.~.:u ~me D+ ia.
lJuough 9. can be present In lhose donon.~.:u ~me D+ In.
dfvlduall h_ve aJlO been ryped II RHD'neptl~ In this M.
Sly. JI8 Pardal D Mants In whlch f.'XOlI4 and 5 are InYOI~
such u 1)" typel ) and 2. would be ryped RHD-nqJluh-e with the inaran 4 ~ wberea, whm emn .. or 7 il In.
voh'ed In pania! D. RHD exon .. and 7 ..~ would 1e8d to faJRconcJusiona. ~rore. at Ie8sctwaRHD-ryp'l1I--.
shouJd be comblned to obrain mb~ reJlabI8 muha. UI.zt
Ewon
thea. dfscrepaudel between 8eJ'11typ6ngandpnotyplna can
. ocxur.Avmt et ""dewloped a multipla RHD-typlnc"'~
bued on the ampWlC8tion ofRHD Inuon .. and the 3" NeR.
WIth thJa muJdp1ex US8J dlscrepanciel occurred In panJaJ D. Malt D. and D- pllenotypa (dCe and dcE). Thua, In tbae
"")'1 d181 arebased on the ampJlfkadon orNeRa. dlsc.-p.
andes OCOIr. In OUt
opInJoD.
DKW reliable RHD aen«KYP1nt orpudIJ D- varianuls buM on IN dJ&rences In the cod- Ini segfoN. becau.se tequenceslD those feIIona nentuaJJy detenn1ne the pepdde.AIIbouab Ilbas ~ 8hown th8t the prnence of RHD 9On l. non 2. pan of exan 3, exon 9. and emn J O does not lad tO expreaJon 01 any of IM preHntly known D epltopes CD-CE-D hybrid Jene. Donor 1077),; al' qualltadwe D varianl8 known at prnnu. ilie IWOdated whh the expreISIon of D epitopeL Therefore. If illmponant 'o estabJUh the pre8eDCe of RHD sequenc:es. bec:ause ther mlgh. Jead tO the upra8fon of dlnk:ally important D ephDp88.Funbermore.1he muJtlpJex PCR pn.J1ted he88.. di.
rected at åx rqion8
al
RNo. COYerina aB f'XDnII wtch RHD- specfIk: sequences In Ihe mdlDl ~ns: this martcedJ)' re.duca the chince of JrfIeradnc falae, and espc:ociaJly faJse-nesatlve. resuJtl, u compared lo Ihe chine. when 5lnsJe-rqIon RHD genoryplnl85S8ya an \Ued. ~ for [)IlA, aD quaJhatlYe RHD vari-nll (total or partiaJ repace.
D1eI11 of an RHD ~son by IlS BBCE equiva1enU IcnoI,vn al present. can be m:opiz.ed bythia n1ultipIPI PCR technlque.
D'1*"caused by lhe repl.cement ofRHDes.on 2 wlrhRHCE exon 2. Because RHD ema 2 il equaJ to RHCE emn Z. RHn exon 2 cannol be spedlicaJty amplUled. When (urutnownJ mutarions do noa fnvo1ve the RHD-spedflc nudeocidft 8' the 3' Md of tbe prim~n used in rhls multipla PCR 8S!I8);
they wiJJ be ~Issed. 1bII me8UI that quaJUatJve D venantll caueed bypoint muladonsaucbaa ()II, 1)"11. [)IMI. r>HJo4I,and
12345'78
-IC
-noaS -exoa
t -IX- -daD
.
CX08,
-aoa6
1 2 200 bp
167 bp 95 bp 100 bp
fil.
S. all8llha 01 Ii'" ASP/&. SpedIIc l)MI product, 81 bp! In_.MI amcrol frepnftII. J t7 bp. L8I8 J. DNA muhrt la8 2, 1>+; l..8D8 S. 1)..: 4.7. IJ'Mr I. w.t. CCIDå'OL
Volurn8 31.
Noven'WJD.M-
1998 TRANBFUSION 1011.
. .
MAASKANT-YAN WlJK ET AL
!)DIll wm be mIa8ed and wUI appear u normal D+ ID tb8 multipla PCR. The four DVII (T329C mutation ID mm 2 of
RJIDU) donors Indeed wve typed In the multiplel PCR . hav1n8
ncrmal n In
thJ8 report. we allo delaibe aD- ASPA.An81ysIa with thJa U8&J
8bowed
a DMr.spedftc PCIl prod- uc:t. whJch tJt the DVII pheDotype In thae tour donom Be- C8UI8 of cWfeIeDt dmJm8tmce8 In the respecdve PCIII. II wu not poa1ble to 1ntept8 the D~ ASPA mm the multi- pla: PCR. IJI* can be IeCOSDJzed becauae of the multipJ8 PCR becauIe of the Iaclt of amplJftcatlon productJi from.RHD eJDDI S 8Dd 7. 'l'he I8V8rI8 prlmera In exona S and 7 are sped8c for thOl8 RHD nucIIJot1de8 tbat have beea
.
placed by RHCB nucI8oddelln the IJI* genotype (M55C and. G1048C, l-.-d~.
Be8uIt8 from the muItIpJa PCR may be confInned
br io-c:aDed
bybrld PCRI. Por aamp1e, we developed a hybdd PCR to conJlrm the D- 4.5.8 COlmlnlon pnotype.lr 'lb8 RHCB GOD 8-1p8C11c fbnfud prlmer 8Dd the RIm 8IDJ17- spedftcmene
prlmer pmerate a productonlywheDRm:8
UDD
,
8Dd RHD UDD 7 are prennt withfn ODe gene. D'"4.S.' convuatOD S8notype8 of DODOlI 509. 570. and 881 were confhm.d with tida hybrid PCI. .
We ha,. appUecl tb1I m~ PCR technJque to am- mode flufd (Ful BHW; et aL manU8Crlpt aubmttb!d). lDd we lI8\I1D8 that II wm be of peat vaIue m pI8Dat8l cUagno- Ila. B881de8 the f8cI: that tbf8 multfp_PCR t8cbn1que la..
and 1ICCU1'I.t8, the ampiee requI.red are amaIIer becauae tb8 mm an.aIyIdI can be perfmmed In ODe reKtloD mf:ItuI8.
1be mIntmai DNA1Dput 18 onlf 10..
In addltioD, muldplaPCR lIWyIta
muled
a UlI",~of D varlantl not lU8p8Cted from l'OutiDe D HIOtypfDc ...
ault8. From one doDor (Donor ISBT 80; C"cee). who W8I typed u D-, RHD exma 9 W8I amplUled. 'l1:da mfght 1mpIy the pr8lIDC8 of the same ~~D hybrid pne u bl DCJJI«
1077. In the poup of 48 DOnwb1te D+ dODOI1, 2 were found to ha.,. D ~ and D". 'I11J8 couId mIlD tbat the frequencJ of D varlantl, 88p8daIIy In Donwbite8, mtght ID filet be hf8ber tbm hu been embII8had by aero1osk: metb- od&lI
.
Becat18e of the Imolwment of aD mina wtfhRRD..--
.- ....
<e
~
lSecauae Dr me ImUmmmt DI au mtona WlUlRHD-ape.
cUIc lequencea
ID the codIJII
reslDDlIn thI8 muJtfpJR PCI 881" new pU'dll D paotyp. and hetmopne!trIIDODI exf8dnt
D pnotype8 can be euDy di8c:overect wlthout the use of 1arp panela of MoAbL BecaUle D varlantl tID be mJsied with rtandard eerolop:methoda
(ODI monoc:loD8l and a polydonal mtI88um) and wfD be detected on1ywbc -appropdate antfbodi.wJI1haw belll1 produced.1CI8eI1Jq with themult1pla:PCRmJgbtbeU88fulfnr IlTm ' -appropnate anUDOa181WIIU1P8 Deen prodUced, lCt'eeI1fJ1I with the muJt1pJBPCIl mJcbt be uI8ful for RIm J!I1OtYpmc ofDNAfram redpleDSI ofb1ood tran~,d0Dl8Dd e&pecfaDy of tbat from presDIDt WOID8D lDdpremenopauaal womea.
'IbIa wouJd strongly Ieduce the cbmce tbat padenll typed u D+. but who In fact expreII I D wrlant, recehI blood from I donor with normal Do
lDd thf8 wouJd thua
Pl8V8Dt and-D product1on In I D-9Irlant mother prepant with a1 020 TRANSFUSION VoUne
38, Nowmbeu'D , 1-
fetul wItI1 DODDIJ D. 'MI tD1DIt a:w m.. anOWQ be coDlid- eradOD ofwhetber It II worthwbJle to type padlDtlln D88d ofblood transfua10DI, espectaIIywomeu In tbeJr cbfJd...
.
I - - -.
- _hu -- '~L ~LI- ___I.. I --(etui
with
. worthwb1le
totn
especIaIIywomeu j
tranafus1onl,
ini
)"8111 and ptelDlDt womm, withtb18 multipla PCI.
Receot!y,
Danle18et 81.18
n!pOrted that J)..~ baw
Intact RHD. Okuda et aL u reported the same for J~neII donon. Undl the molec:uJar bIcqround oftheee1Ddtvldu- aJa la knDwII. every PCR
auaywm pv.
faI8e- poattIve rtJ8Uhaamonc
tbeIe populatlOD poupI, typm.,I>- h1d1vIduaJawbb
Intld RIm u 1>+. However. Ii ahould be emphulzed-
88 oUr
,wm be
mostly appUed to ttmd.Is1on II!CIpIEl and In pnmatal gmotypJnr, tbe cIID1cal conaequIDC8I ola faJJe-p0atdv8 result are le8188V8r8 thm tho8e of a faIIe.neptM l8IUIt.
A reUable multiplu PCB , ta pNI81ted, In wbk:h
aD uona with RHD-speclfk: 88qU8DC8l1n the c:ocHDI-- glaDa 118 aDaJyzed In ona 1'e8dIon mIxture. By 1he 118 of ddI PCli
... the
0CCUI1'IIDC8 of dt8c:repancte8 betw88D pbenotypfns and genotyplDa r8IUIt8 wfD. be reduced. '1b8 q1.iaJ11adve RHD varfantaJ)lllt, U-, [)M, D., D", U-, 1)8', aridR. .. cm
be recognfzed with tb18 PCB.ACKNOWlEDGIENT8
"lM wman tb8DJt P.c. IqtIwt. CLB Am8t8rdam, for eIp8It
~
ce;1ft.,.,
p8I8fW ID HA. ZoDdena, MI),PbD, DIpu1mIDtI of Ob8c81dc8 md
~.
.A~""M'" Cabr, AmItIrdam. Jbr 8IIDdIDt b1aod l8JI1pIeI; l1Id tbmk Prof. c.P. RllpIf\:to8t lind Prat D. JIao8, al Am8I8rdaa, lJr cddc8l188dJDa of die III. n.~
REFERENCEB
1.
C2Mdf-ZIbar B, MaUII WO, Le VID 11m C. et 81. I.oc:aIDdon of tU human Rh blood poup ... mucturllD ~
1DOIOID81pSU.lp31.1 repD by ID dtubJb~
au. 0IaIt l.l~ tOO.
2. SmJdI8 11. AWDt HD, Jud8cm JA. et 81. ~..wa 01 BlID lDd BIlCB PD8 producr.w
'*'
ntroml tr8DICfw:daa01 I!G ceDllI1abum. the moIee-lIu ~ allh
bIDoCI pouplDtIiIIIL
BIoocII998;87:2888-7I.S. Le VID
Ib C. a-tf.Z8Iw
B, RaJD8IV, et 11. ~ Ja
'!IIII fIpr lINA 1Iobm8 118 pmcIuaId
by alt8maIh'8
tpIID- JDa. BIood 11182;80:10740-8... Co. Y, a.tI-Z8har B, Le VIn 11m C. et 11 GeD8dc
b8III
olth8 BhD-poIIdft lDd BhD-Depdft blood poup pI1lt- morpht8m Udet8rmIud
by SoudI8manaIJIIL ...
188l,"7I:27C7-51.
&.
HrImd
CA. W~ Le, SaulA. 'lbree UIU'8Iat8d Bb D,..
polymOlpbllma
IdentIhd lmOIII blood doDmI..
Rh8IUICCee
Cfr') pMnotypeLB1ood
191N;N:S21-4.I. UDIIDIIbI P,
