PCR Artiklar 2 metoduppsättning

FY GINOTYPlNGlY PCA-ASP
..
iii
u IIUJIIor DNA II'Omfrom
penpnem
01000
CIIUL
pertphen1
blood
cellL
M
:
711
419
DlSCUSSION
TheFYbJoodgroUpsystemfl polymorphlc In lPOItpapUa
In
603 I&tIODl,
with \WOaDel-. lY and~,1npeople
olBuropean
310
andAllan orf8in and I thJrdalleJe.~ wh1cbpnxIUC8I no
fy- or .". on red ce1l8. ID people of Alrfcan orlsm.14..1 We
310
~1'1' Fy,.,..Ft'fIY'
...AJ.
Jo ..,r--Fl.. 2.
2. RIp~
alp."""
lY pnotyplnl
8bowta8the..
pi ~
pi
pi*'~
p8t18m8 0""-
FY
FYpnotyplac
pnotyplac pat18ml
patWnl o...,.
0""-
ha" dewlaped I FY-genotyplnctest bued aD the LIM of
PCR-ASP.
The forward primen are speciftc elther for the
seq\1lDce of the ~ llleIe or tor that of thelY o r PIaDel. In
the S' noncodini region of the FY gene, and the reverse
primen areelther lpeclJlcfor thel8quence of thePI aUeJe
or for Ihat of the FY"or Pyalle1eIn the codlnl restonol the
.60wb4the.-
la ddllCUdJ. G8aoiJp88 ID
la ddllIUdy. G8IGtJP8lIa
berlpchandp8II8Ilnvolve
the..., ", 81.. ud ,... enthe
Py uWe ud W8n
therlpch8nd
rt..uh8Dd p8M1lnvolw
,... InYOMthe""'l
d
DUllIeI'8d In -pa.
h.. bI8dISouda
MrlC8D
don.. only.
c:uuntend
AfrIca cIonononly.
countend In
In -,..
lImP" rro.
hin bI8dI
bl Souda
lYpbenolJpe(abow)aIMIFYpnoCyp8(beiow) de8lpa'"
FYpbenolJP8(abow)aM FYpnorype(beJow)
818del"'.'"
IN' 88dl1U1pI8
C818pry.
Pour
I
,
pie... lIIowa,
ror88ChlU1pI8C8t8p1'y.
PourW. ... ...,.. an 1Ih0WB,
D I_Table
2).2).
MM
corr..pondlJlllOp~
ma. B,
A.C,
B,lIId
C.8Dd
D (- Tab18
epreI88l8 the 1IIO1cuIa'"
muar
ll8ID
repr8I8d8dMlIIOIecuI8r...
18" ~l7tBP
~ 7tUDNA
DNA
II81II
Ull Tedu,-""
G81tJI8nbarJ,HD).ArrowIto the... de(Ul8
T~
Gllt118r8bar8.
MD).
Am",
10
the
IletIM.. oflb8..pUW DNArnp.a. hm th8 ABa18ftdepietlb8 ål oIlb8 _plUW DNA,.,
from theA8O
.Ut-bp)
ud Pr (711-bp)
UIOW8ID
dl. rtpt ... the
(tll-bp) ud Pr (7l1-bp)
amJW8 ta th. rtpt
~..
--
gene.Th, telt ts reltablt androbust,IIvtDllOO-percent
toprllMr
ID""'"
bp)bp)
aI...altbe
~(~.. ...(ID(ID
al
af -- --
concordancewith I8lOlotPcallydetermlned pbenotypeL
PmIolllly dllCrlbed metbodJror FY pnotypinl haveInvolYedthe UIe of the r8IIrIctIonendonucleuee BanJand
Sl)tL M ..., Th. medlad we haw delCrlbed ts more economil:al,1- tfme.cof1lUJD.in&and euter to perform:lt re-
... dM
DNA"
DNA
rr.m-...
quin8onlyPeR.with DOdJtudODst8pi JtrequJr8DODpen8iv8 nIIIt11ct1oD eozym.j
carrfed out la lO-J1L~1II.
and the ampl1ftcadoDl cm be
whJch reducee the qum1Ity
CIllndlcatedhetemzyJoåtyforthelY a11eI&TheM8k PI'
PCRIndlcatedhetemzyaoåtyfor the1Y aIleI& Thew.t lY'
,n
wu coDIrmId byweakreactkm8
apIDIt apaDå
9pI'8I8Ion WII COD8rm8dby weakreactkm8 apIDIt a panel
,fcWrenrntand-~ reapntland Wo by ablOrptIon-and-
conawned.Theproceduretab8 1- than 2.5
boun from start to l8IUIt. adudJnl the DNA prepuatlon
Itep.'Ib8 Ull ofheat-actlvatedDNApoJymerue(AmpU'Dlq
GolcDprevent8tm.posJdYI r-=dona bymhibltlquupe-
lOdaD studJeL ntradoDl
c11IcmJsprimlnl
of retpnu
ofdUrermt antl-Pf reqentland
Wo by ablOrptIon-and-
with tbeIe ceII8 and poIydonal
or
elutlon atud1eLntradoDl with tbe8eceII8aod poIydonal or
10noclonaJ
and-Py3cUdnot Ibow signUlcmdylow8r tltrImonoclona1anti. PySdJdnot Ibow sipliftcmtly loww titta:00KOnt thaDwer8seenwith control ceDa.Phenotype
tion KOrM tban we18 seen with control ceDa.Phenotype
Delpmotypl correIaUona
andtheca1cuIated
pmetmquenand IPIDOtypecarreIatIoDaand the cak:uIated pne fnlquenI. are aummarlzedJa~1e 3.
ct. are .wnmarfzed In nble 3.
M expected,primermixA, madeto det8Cta bypothedM expect8d.primermJxA, made tOdetlCt. hypotbedIl,ca1,
sUent,
recomblnantaDelecarryll1I both the GATA-l
.Ilent. recomblnant allela carryll1l both the GATA-l
aodf-d1arupdf1l
mutation and the Fy"poIymorphJamat
motlf-dJarupllnl mutation and the Fy" poIymorphJam at
ucllOt1de
131
('JW)Je
2 andPt..l), D8V81'
yteJded8positIve
nucleot1de 131(1ible2 and Ft..l). n8V8ryIe1dedI positive
Iactlon
with
the
samplelltUd1ed
by
01.
Und! sucb an alreactlon with the sImples ItUcfied by UI. Unt1llUcb an al~eleJe
JadeftDed.
thJI
primer
mJJ
elther
can
aerveu I nepII deflDed. thII primer mil elther can serve u a nesavethe
controllor
unapecUlc
ampUftcadoD
of the.
FYFY
pRe
conttollor UDlpecUlcampUftcatlon
of the
pRe
roduc:tI
or can
IJmply
be be
omJtted.
DNA
obtatned
horn
productJ
or can
slmply
omltted.
DNA
obtalned
horn
nnJodc
tJuJd
pwsave
l'88uJu
81dear
andand
eully
lnterpreted
amnJodc
lJuid
re8uJtI
u dear
eully
interpreted
.
durlnl dut 1D1dII ph... of PeR.
PbIDOtypeand pol frequend. obtaJnedfrom the
100wblteSwed18h
lubJectI ""M qu1tec10181y
with thON
determJned
from pur8ly8OIop: anl1yllJ of red ceJla
from
BuropeIDIandwbtteNa1hAma1caD8.11PrpaefreqUIDd.ln peop1801Mrtcan ortpJ aremorevutab1e.
Fy18receumtoJYand~,
and10 lt II not poIIible to detmDIDe
aUe1e
Crequeocl. d1rectIyfrom 88I'OJopct88tl. Prom 100
bl8ckSoutbAlr1c:an
donom,
weroundthatPyb
rr.
qull1CJof 0.798.Of the 159Fy,n.. detected.aJ)bad th8
codoRrorAlp at poaIdon44.theHquenceencodlnt for PI:
no PyllleleencodlQ thellqU1DC8for fy- WlllouncL whk:h
,uppom prevfOUlOblllVldooa...t.., No ~ allel. wu detected1Jn0DIrh, 100wIJlt81.
Of the 200 dOnoR telt8d, rhree bad wealc exprMIlon
01
Py'. 1\w of the thr8e wue Swedish
..
TA.Lls. PhInotype.
genoIyp8.
andIII'"
fl'8qlMftcIM
Inthe~...
8ouIh
Afltan
graupe,
. del8rmlned
SW8dI8hand the bI8cti
pnuntld PCR-AIPFY~
lN8hod
PtwIotypI~)
BInoIype('%)
AIIII ("t
8Itak Wtft8
Blå
WtItI8
BIa.
WtIII
~"')
:yc8+b+)
4
2
:YC.-b+)31"
21
Fy'F)4
O
21
FY-
<4Ot
f'Y"P1
~
4
2
o
4Ot
fY
Fy
83
O
3t
Fy'to/'
PI'F'1
:y(e-b-) 113
o
PtFY
, lncb8I CN Fl"'"
W88IdW~
~I
two
w.
I
..
fAl.
.
o
a
41
'7.r!llt
78.8
o
IYPI pnotype and
rheOtherwal
. blactMrlca
withthe
D
wbJtee with the
JY~genotype. 'l11.eW8akPf and.
B8I1Imayrepl'8l8ntthephenotype
caUedFf, ..,11 but,81themoleculv
b8818ofl'f hu norbeendetennlned,
P'I'
could
be
heterolenooua
and
_ol
d reprel8ntanywelt expreuJon
alP-/'. Mall/naoner aJ.'round no devfadonafromrh,conaenlUl/ypno
\."uu&
sequence
whenInvesrlptlr1lrwuIn.
dJvlduall carrylng /O)"gona. Weak-
V.me 38. FIIJMIy 1-
TRANSFUSION
171
OLI8QIIIT
AL
REFERENCES
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IdeDltJIca.
tIon oflll eryduocyt8 componeat curytnl the Du", b1ooct
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2. l1t.uIht.1'tA. ZbIZ8lll8 V, JohNon C. et al Purl8cadanlDd
~
of ID 8rJduoc:yIImembnn8 pnIIIIn compta curylnl Duft'yblood poup andpnJctty.PoIIIbIe...
C8p1llr
m PItIunodhun
IIII!II and PIIIsmodlum
ma1ad8
puultL J BIaI01Im 198'.284:13770-4.
3. CIauAud A. PoIyåDvaJ 1.hn8m8Y, et II. 00nJncof ..,.
copnllllln D cDNA. which encoclnlhe nJar IUbunlt of the
Dulrybloodpoup Iystem Uld the receptor ror the Plarmodlum låtIx maIu18paul..
Proc Ned MId Sd U SA
1993;11:10795-7.
.. Wualawlb I. HadIeyTJ.TheDuft'ybJoodFtUP and.ID updIae.TI'IIIIfuI Med 11ft 1994;8:281.&
QI,
5. ,.1'f A. PoI}'llrova J. Zbrzema V. PcwIAO.ThecodiDll8pJIDC801DuIfy bIoodPOUPpn8 IDbWIWIIand
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len'" polymorphl8a,II1IIbodyUIdmaIarIaIparulUI spedlcldeI,and~
In
lIOD8IJIbmtd
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1995;8:815.21.
I. TOll-m.. C.Le Vm 11mC.Gue P. et al MolecuJub8III
bo...,
I
lOd PCI-DNA typJJ1Iof tba lyA/Fy" blood poup potymor.
sultable for tbJafuDcdon.
pbl8a.HumGenet 1995;95:"-10.
7. lWUIIIIIDSo0mI T. ~ B, Ibmatu S.GIaom1corpnJzatian afd1egIycoproteID
D
DuJI'ybloodpIUI) ""'1"1aDo-'1JII8ID II t.~
with poIymorpbiImat
.. shown by the IIIUIt8 of te8tI on
.
DNA derlv8d from amnfocyta
BecauIe a f8tua with W8It
PyI' would belnterpreted II beJnc Py(b+), wbIc:h couIdraI8e
the 4HmJno KId r8IIdu8. BIood 1!ll5;85:8Z2-&
8. N-IItG. Soo a, Sc:hd TJ. et aJ.MuWlona In the ayth-
unneceaary CODClm for bemolyUc dJ8eue of the newbom,
the 18UIta of retal pnotypq
need to be Intmpntld In Ch8
rocyIII cIIemoIdDe
r8CIIptOr(DuI'y) l1li8: die moJecuIu H" Idel.'ttfLo.ttnq
of a cIeIIdoDID
contat of otblr tndfc:e8
of t'8talb8molyII&M polntl!d out
III oItbI.".'Py"an
by Mwpby_11.,17mIItyplq ofblood lampl. with. WIIIt
PyItcu eutJyOCcul,
dependlnton ~. and-~ reapnt
~(~
uaed.AltlJouF
the dInka1"p'lft~nC8 of traDIfu8InsID Fy
(b+ ~ bloodun1tby mJ8taketo . patientwttb ID and- P'/
hu DOt buD 1lUb1tlbed,the rl8k for sec:ondary
Immun1:lAdan and ID lCC8IInted c1euanc:eoftranlfu8edceDacannot be ududed. If trulJ{JtnIdcally) Py(b-) blood 1818quJred. tbeD pDOmk: typins of thePY1ocua
In blooddonon
the
DaIf
... 01an appIlIOdy
b8Ithy
Indfvtdual
wtth tb8
Py(..+J pbeDotype. Hr 1 Hum8toII995;90:82S.1.
9. IwuaoIDS.U 1.0mJ T, et Illctendbdon
of. uonIlIDD
and Ipbd (mm ofDuffy mINA tb8t 18tba predomJnaDt
tI8DIaIpt In bodt erytbroid and poItcaptllaly ".1118 IIIdcJtb8bI. 1UoocI19ll8;87:37H5.
10. Neo_r. M_TY. lCoIUawUILP lr, Scb8I111.PuDCdanI1
md bb:berulicfl anaJyiJI oltbe cloned DufyandlIB
JacertalnJy ID optfoa.
Id_.
UtJ wtdI the red bJood ceD chamoktne rec:lptor. BIood
19M;I8:44-52.
Il.
CbaudlartA. Zbaema V. PoIyUova1.et Il ExpI8lan
tb8DuIPf antlpn
ThI8 coJlabondon wouJdnot haV8been pouiblt wtthout the
CDn tfnUOUl support 01Prole8or Dmd J. Anat.
. the
1n18m&-
ttonaI BIood GIoup RåeDc:8 Labcn1Dry (Brl8toL Ut) lUr
I
wbtch MLO II *' mucb!Ddebc8d.
BUzabecb
Smert81theNatI!
BJoodTruIJfu8IonServk:e,
PID8towa,
SouthAtttca,II thuIuId
mlldnrl ndabJe
la lS82 ceUl. Bvldenca that It 18the bu.
l1I8D8IJIbrocyI8cbemotInerecaptDr.lBIoICbem
AC KHOWLIDQMINTI
b
bIood 1Imp1. from South AfdC8Ddonan.
1994;287835-1.
12. DvbcaI8 wr. lUceGe, MobJerMA.et U. Redblood«:6
III . .. rorIDtmieukfa8, altutoc:yte chemotuln. JCln
lDV8It1.1;81:1382.9.
13. 0\ttnI8 CH,ClludhurJ A. Horut Roet U. 1bI dolDllDon th8
DulI'JbIDod
poupIDdpDforblDdJntPIIattIdIum.....
lIId P.btowIIIt maIWJ puultel ta eryduac:vteI.Jl!Ip
Med 18;184:1531.8,
112
o,
TAANlFU8ION VoU'nI 38, FIbn8y 11181
FYGINOTYPING lY PCR-AIP
14. Iultt PD.ApplJ8clbloodPOUP..roIOlY. Miami: Mon.,mtry Sc:Ientlftc PubllcadoD8, 11185.
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1995.
II. Mou1dIJM.HayeISo W.u. TD. N8WDuJfJ
lfHUIfnqU8Ddll
In blKb U d8t8rmID8d
by PQ-RPLP (ab8tr8ct).TranIlualon 1991;!8(suppij:48S.
17. Murpby Mr. TempllltoaU. FI.mlnl J. et al. Compertlon 01
II.
Py(b) 8t&tuI u d8t8nnln8d mop:.uy
and pnedceBy.
TranIAaIMICI1997j7:13S-41.
TOWDImlUt C.CoIIn Y.Cartron JP.1.8Van Dm C. DUrup-
tion of a GATAmodf In the DuIfy lene promot8r a~
erythold pRe apn8icJIIln euay.nepöw lndJvlduda.Nat
. q.
Genit 1985;10'.224-8.
lwunoco
S.UJ. SuatmolO
N. ICal OI8nctartzadon 01rh.
Dufry pne plOmo
1Vld8nc:. ror d81u.-spec:Ulc
abolltb-
ment of apnIIIon In ~)
ofbl8clt lndlvfdual8.
BIoc:bemBIophyI ReaCoIIUIIUD 1995;222:852-8.
20.
tnD8CrIpdoor.:taraIn bemato20. Oddn SH.GATA-bmcIIJII
poledc caDa._ODd lH2;80:a75-8L
.J. MUItrSA,Dyt8IDD.,..,
~
HP.A Ifmple
out pMCldwe for aA...m.. DNA hm human nw:leatld ~
Nuclek: Add8 Rel19l8;18:1215.
22. SambruokJ.Pdt8cbBP.MmIItI8 T. MoJlCIIIar cIoaJn..
A
Iabontoly lD8IIU8I.2nd ed.CGIdSprint Hubor, HY:CoIcI
SPrinllivbor Pr-. 118.
U. Avent HD. M8rda PG.Kel typInt by l11e1e-epedftc
PCI
(ASP).ar J Humatot 119l;9S:72NO.
24. Ma1JIuoD
Go au..
°,Kå
So
et al.A CauClllu Indl.
vldU81wttb lb8 Py(...o-) pbIaotype bu
. DDY8IdaIec:tin
the FYpne (abInctI. TrIDIb Med 1998;5(Suppl):11.
AUTHOR8
Martin L Qtuon. PhD.MO.StalPhyatclan,BIoodCenflr. Uni.
verlfty HoapItaI.5-22185 Lund.SwIdm Ut.pllnt reqUUIII
CuoIa H8J18IOII.
8Sc,l88I'Cb Stud.,t, BIoocICenta'.UnIVIIfIIIIV
y,nUy HOInltaL
HOIpltaL
N.U D. Aveat, BSe,PhD,Mu
, MoI8cuI8rGenedc8Seetlon.latemadonll BIoodGroupI18ference
Laborahxy,BrlltaI.
~
lnpIa 8.Ak8oa. SIInIorMtdbI TecbnoJoaW.BIoodCenter. lJDMntty HoIptIId.
-
-
~
c.,.
n
A. GNID. PlBMS. a..rcb
n_n-..
Sde'1dIt.
-__n.
BrIItoIlnIdtuta
ror'1'raa8taIIon
SdInceI, IrII&å, UJ:.
~
GeoffL.~
BSc.PhD,SIllIorR-.di
1brTranaluåonSel_ca
VoIIIn8 38, FebMIy 1988
PeDow.BrI8toI
TRANIPU8M)N 171
~
.-
t
~~
....
RAPID COMMUMCA 'nON
-
Molecalar Ba.l. 01
01tbl
the -eli
Kell ,(Kl)I.J ...ue.v~J"'T
By
R81d,
BySooN8 LM. Xu Wu. Marlon "-Id
., (I.~ I..
~~--.a
I1811;
.. _.8 - -
"'-'.1 . fil 11.1
- ..1.111'..
III- . ..IQIICIDNA8
J.II. N' II_. ~
1"""""'"
II8UIt
.. .....
,
fil.. ft8'I It.. 181
.111.""-'
h8m8IyIIa
nIn8
~
... 1['la.
fil 1MW
.. Ad .III - L-1Id.A..
81
MIll,
8 C te T ....
t8
...
-
-
-
fil
........
1- T18II...........
818
II _Me."'"
X. 11Irt8M III "~I\.
8Iv88..b...
.. ~
..,. C.. T
no
......
8811
Z8IInlld,Iftd CoMnRedm8n
lA8L
..
...
I
- 11--.. -II ~" 181""""
fil__11M
. r_""" ..,..
III41.....
~f Ildl. ... 1111
t 1['.
te""""""'"
"""'4
IIRI 1181111
..
III.. fil tI88111,"111."
.. _.tIL ..
1(1
..natHl6-"""'". _.- 1 1IUtUJ-.11/11, UI
th8 ~
111'8'1 a (lit""""
w
~~.,.~
th8
th8
",.~""'II
L-
IIIJII. N"""
th8,. -
*1181
8M tI88~
. I 1111111..
. . IIIIII"1
l8M8I...
,
THB
A
18""
~ytic
IQ, ... ..
1181""""
ni..'"
fil th8.11fl118O1VRL-",Ilh..L
... ..........
fil...... ... (aufil. I""
II
r\18IIII
,..,.
DNA'"
P.I .. I k ...
..
Or- 6r n. --11M
01
uw..
.
III
.. 1181
fil '18n'!lld..,.
MoIecuI8'cJoaiqell8bU8bed
dIIIIKel! bloocl~
-IN C8I'ried
OD. 93-tD typ8 D aIY "" Je Kål
proIDiDh8 . lIIIJIt, 46 lIIDiDolåd. N-termia8IdoaIIiai8
die ~
lIId
C-fIIInDiD8I
pordoa.
01665.......
...
.
.
add8, OD die WInIaI..ta::e 01die RBC. AD of die c:artdydr8eI Me N-IIabcI' I lIId are prob8bIy 10C8IIId
la,
K3 (Ep-). ICA (Kp"). lIId IUI (Kp");
Ull
,
"
samplel. Amoaabiocid JI'O'IIII8Im.I'-wd by 1IJoi...M_..I...
1108, Itl (It) il pobebIJ 8DIII onIy ID D(D) la ~ RII
sy"'"
U ID i
,
tb8
di8eue ID d8Wbon
". .KeDsyIf8m 18
compIa II1II l'DIII8l1l8I 20 diIffnaI ~reI88d -a-baw... id88d8ed.
K8U
...,... ID be l!I8IXIdId la
II...
alllditbedcal p8ind aDeIe8
Idp. ad
low-prevaJlace1IId
'IbuI. Kl (K) ad K2 (t) ... prodLICt8
KUli h 11"~"jII"
t' r.. III fil I 1~
8
. R 1111. c...
BLOOD GROUP sylfem il ~
iD
Mfjba Ilet ca c.f1I8dicx.ID lI'8II8Iu8iCI8al i8c"..Ic'"
bIood ....
KEU.
lnDItuioa
~. --
L
K6 (J., II1II K7 (JI'); KI7 ad KIl; 8l1li lU4 ... 1t14.
However.a 11UJDber
al ~
- . ! . lUdI .
KJ2. KI3, KlI. ud K22. Ull indDpelldeallye~
Ibae ret
lIId th* pl8ce in the KeII syItaI ba...
beea
w-t
ltII'ouPthe )'18ft ." ..-oIoP; lDIIyI8 al
iDformaåw rlmi1ie8.14A reccndJ
developedim a"p:
IDC8OCIoaIaadbody-lp8Ciic
i hIH,..u., al ~
upuaaiDel93.
s1Udie8MI"""
"'. \91,34.5.
ud 6%7.
BartybioriIeabc:8I
dill KeD
N8Id808 . proIIiII
whole c:oaIormadma
la Iuply dl!pt""
,.
boad&. 12
011
cIiauJftdII
lCeJIproIIIia- 16C)'II8i8 -d18.
1 in die er..rep. lIId I' iD ilie
portk& Je Redacdaa
ofUCIbysuUhydryl
paa...
iD- 01K8D
--
u8CHpi~
12.1)
rocyt8
(MAJBA),
.- 1IIODOC1oa8I_b0die8 The rnoIecuJ8r'-il 01 die cIift'8ea&KeD ~
11M
o cIift'err8
KeD.-I... iDd~ - cIifti:raII
-paa
Dat ...
de8armiIIed.EqnI8toa 01 K.rI pnIIIIa la
)cc...ID..,..wJydiIdDCt "'0lIl al cbe~
'Ibaa.
traDIfect.edceD8UIiD8. wild-type
cONA"'"
la IM
I(IIK2 andK6iX7Ull dOM 10""'.
KJlK4epitope
detectioa01 the biJb-prevaJeace
JC2. 1t7,
Kl.
. in a diI'feNnt loc:8do8 8l1li KIl 18in yeI anodI8r pro88åI
bill not01... Iow-prn"'"
-l1li8
Kl or KJ.I) 11å
10II18in.'lDMritIDce188IIIiD8OmIII
ad c:odomiMIIIandKEL
ftndiq i"""'"
thai leV"" 8l1li..- 1118)'
be curi8d OD.
...
m8pp8d CD,~0ID080ftI8
71133.'"
Ii.
proI8UIandtb8Ilow-prevllence-.prob8bIyreexpoIUN
0110II1II
...
An utibody CDIt I. named 1C81aftIr 118pI'OIICIIitul, lada
with lppI'Oaim88eIy
9* al ra8IDm red b&oodc:cII (RBC)
...
side ODvlrilac procaina.Haviq l1Udted
lI8IUC:IunI 01...
KelI ...
1dead8ed
die 19 aoII8 dill ea:ode lCeIIpro-
tela.1.we have now "".Aiood
1',."
lIItIoII
. LI1rtbII1I'.
Otrt8IIIIIMJrt:It
~
tf.
New Ycri
c.ar. N.. fort. NY:.. ... Ifj lÄc ..." ~
UrtMrrIq tf
~
c.-..
$"""'" C1c8*r 17. 19H:~
MrUUeM Id, 19M
~ J.r J rMI III ,."
,..." I
tf "...
c;,.. No.
HLJJMI MIll ",. SMrr'o.. 11ft, (III CL1 MIll lDlCO,.
No.ItIAJJfJI
- '" ~',
.,
..".,
~"tJa
"11"01...
(III
r.L).
AMrwa ,."." ,
", tÄlt1III
PItD.111Newr "'*
lIItIoII c..r. JIt, . 67 sr. 1hw rort. Nr 10I1Z1.
7"- pIIIJIftIIM aM8 tf tIfI6"'*'- w,. ..".,.
III ptIIf ", "".
c""'" "."".,.. nu
"~I'
~
d' .. axlfJ,,-"
--
wifillI
"..
u.s.c
"* j8:I.
o ItJIJS
'" 111
SocWt7
t( H--.
OOtJtJ...491/~J$J.OOAJ
"2
DNA ud
stillldo8 iii
tf"~
.,
die moIecuI8rb8i8 01 die
KIIK2poIymoqäIm
by ~
"" -.,., "."..
1714""'.
theemDI of Kllltl
dIemIDK2IK28CIqUIIIC88.
A b888...
~
eKOII
6 of tbe K IJItJ paocype predic:CI
.
.. lIIJåIo
--
8cld chuIp. 11IU"- IUbttiIUåGl creafe8 ~
ZYI1le
lite dill wu t8IIId ia more lb8I 40 diffeNa& l8IIIpII8
10 c:oD8mIdill lh8b888
subIåIudCIa
ideDtift.... Kllltl
aeDOtype.
_u.
UIWI "
MA1tRIALSAND MITHOO8
. ln88
.
~A"""'"
~
......
IIIDDII
6'18 42 ,...
.1aI W ~
baw ... ...
,.
DNA WII
"'- I ID5 .. 01
wI8oII
CIIIIIIc8IIIwtdI '-""""'"
ar,
IUICI...
reI8IIWd.., c:8IIII:ifIIpda 1.000,
rar 10"'"
lnI8 ...
.
..
..
.
buI'y COlt.la boIIIc-.
ptOCIdI8Il1dI8atbed .., JaII8
lICaI'l III
DNA
lII8d.Sew801- DNA"""" ...
... Y8I
.. Ne. I,*-, ,ta.,.. PIt81M18
ICILL
\1(1 I
"1
JIt4INO1VPI
.
u.A.JIIw. IM
. ,...-,..-
..
,.
dIIatbAi ~..
Id~
......
... dill d8
L
(#'C-J,
T.. DNAjICII)..- ... cIH17I
... ~
(M8IIII8,WI).X..QII.. ta. ~
. coli: -4 .- C8III, ... ...
... h8
ra...... ........
--L.Ir
(~
,
..
MD).T4 DNA
.... dill
Pr
(88wdJ.
MA). ~
,...
.- ........ ... ... I lra8 New s
lIb88i8Idf
81Dad C'
OdwrNI""
at dIe8I
EJ.. lC2. dI88I ta.
...
n
-
. .
~....
. .
MA~
72"C
-'\ CA). TII8 DNA
Illa (1"
DItW"
..
--
~
...
.. ta. NIm88 (JIII6a8. WI) ..
()då Spill CaI88 (o.~ -y.--)
far DNA r-il &t'~ ...
rra.
UIII. IN).
bIIa8 (8) pII8IIiII
.
RlIUL Yl
c,.",..,..
oJ~1 aM n DNA
NiDIplin
all'anrlld Ind mene priDMn ... U88dID..,uty die 19
KBL ac8. Tb8
al die prima'I ud die ....
ao8
--
ini
... Kl ~
iii T8b18I. n
wde
... ... u
DNA from. boamyl1li DNA. ID aDC88,
ID 1-' kb .....
la.. fram
O.'"
pIOdDcII
obIaåIId(fia I). Al 011188PCR pIOCIucu
ud ~
Kl DNA
... .,-r.d
w1* dill of Ja DNA.MI.at n. -r- ,. 01
eDCOcIecI ideadc8l1IIIi8o
addI80.. olJa DNA.
escept. ~
e
dl88l1a
. PCIt
pradIIaI
- ....
HOIII , ad 6. la ddI PCRprocIDcc.
wIdd8..
iD
.-,
~
lI8IdIDd
sc..In
iDa
...( , 8&:dI
.. lJI8
. QIdck.. .sp.
. L ~.., ...
mo) coIIaa.
DNA ...
.. JIIIriW
,
-
~
.. -Il."'-1tJ
1.2.0:AppW
B~,
."'.,I
~
tbI8I PCR
-
far ...
" DNA
...--
~L
ro.r
0,.
(MadII373A.
V.....
CA).
a- I ..
Tb8 PCR
dII8::dJID dI8
(10 pI.)
... opda88d
I
..,
:2pI. al 35 ",
MA.
I pI. al
10 IIIIIIIIUL - '-,111I~
I. 10 lI8IIIIL I71T. I "a. allOX aI 1NftIr. 4 ,L al -.
... :z IL al , UIIIL ... L '--1ooJ8
...to..Md
65"C. Tb8 DNA .. IM l8IXIo8
mIAtIn
lII8IJ8d ltJ
.. o.a. .......
...
.
,. 90--
su""I""1.J8.
~
~-
I'~
C""'"
I
4
.
oJlelln poIyrrtD"".,.", B- J tIIfIIIpU.
l'eIIIk1ia8
..,... ..
cbaqD
tbaJeI paotype. dUarep. W8I lID8IyDId
iD 41
The C ID T 1Ub8dblda8
c:n88
witlllp8Ci8dty
f~ ..
Idooootil'-
.
L ToCODfIrm tbatdUa...
~--T8III8
--
TCC
TCC
GMn:ACIC1'
etT ,..""
I'.QC TRI CIC1'CCAGAGNIf TCCCAT..,
I'-GM GCITCIG8aN: CM Mlf 11M QM.a'
I'-IICA CICIG
TTTCICIACICACITCATG~
1'.mNlfCCTCACTCCCATGCT~'"
I'.TAT CACICA OGTcm:: eR: TCT~
I'-ATATTCca: N:C TCCCCACACC'TCW'
,'-AX 1M: GGTGCTCAGGCTCT'CC'J'C.3'
I
(1')
ID1IIIIbioniM
c:b8a8I
81
..
"
,
.~
740 bp
eytD8iM
(C) ID lbymi88
.
N-aI)tcoIyladoe.. (Aa. X. 11Ir).T1dI cIIft'w.
eacebetweeaKl lIId lt2 occun18... 6 <PIt2). Bec88
dill
!bl oa1ydUfcnace bctweIIIaJeI
lC2--w..
ch8a8eI la lIIDIDo~
II ... nid- dieb eaaiDe
10
medaIc8i..cbaqe wouId ~
N-8IJcoayl8lDa81.....
&iDe191iD pro8IIiDa
upreuiDI K l, tbu8 ~
IM DI
JU polymarphianL
.- -
fICII
daIre
.. .
~..Å
.A
- IIIPCII-
" a.
lA
P, ~ 4
1.1
0.74
"-QGAACICAM OGAGTGAGGN:r AA/W'
7,I, .
U
11
1.2
t1
1.8
12,11.'" 1.
U
14.,11,'1, 17,11
1.1
1"
UI
I'.TQCI CAT CCA TOG TAC eR: ATG CJM.a'
.
I'-GAGGCTTTTGM N% CCAGGATQA.I'
... TTC ca:
7
NJIC CAC CTO CCA TCT CA T..,
I'-ca: TTTTCCMG GCIT
CAGMG C'TCW'
I'-GGGI eTT ATT TGA ca: CCA GM TCT..,
.
1'.a:T MT ca: TOGAM etT GCCTUT..,
I' ~
.
TOA GGA CAT CTO CM AM AOIW'
I'.TCC
nITGGA
ca: TCC
ca: TreM T.,
-- IIdiefaN8Id
I'.GCJCICG8 Ma
'Ole ini
IN naIIndiegIII8-
...
,.
. PeR
"
--
CCA Mlf
GCC NJIC m
- ...
- --
8ddIdIDer.- ..
&an2
dill eldnaI--
..
~
T.,
II...
... ..
lir-~.
PCII
"
-
. dieI' ...1(PCR
tJI
, tJIPCR
.
,...
" .. 11(PCR
. ....
(PeR
1- 2t.M
-. ,.-
1IfIn*. TII82 G
TII8PCR
tJI
COW8Nd
lir PCII7.
".
LIlITAL
KIIK2hctcrotyaoca
(I... 6 lIDd1) CM be cuily diJUisbed from !ho Kl hoInozypMa becaulCdie 74C).bpb8nd
ils !Ip"t procIucuof ~
of K 1/10 i, men prunounced
_2m'"
"
.
.
. ~7-
nPl'"UAlu""'7
.-
...,.,..- I"'t'""'-,..
n.n-
L -- .L - ", ---
phenoIypeand lllliboda to K I In developedin about,..
of perIC8 ~vlJII . MaJeuDit of incomp8tible bIood."
IfemoI1tk: &JåeIIIe of Ilie IICWhonI(HDN) i. uawdly 1IIIJCi.
lIICdwidl rnIICnI8IaJloIrnmuIIizadon
to IUl(D). buaKl inc:olllpllilhlida c:8It aJsoCMIIC SCYeft HDN....J.Detcnaå8-
4 1.6 Kb
tion of die moIecu.. buil o( tlle KIIK2 poIymorphåvol
will allow fI8OCYPinI
from DNA AIUIIpIaand!dxluld prove
18fu1 iii ~
di8po8h
The
pm""
lCeUpMnoIype ja K:-I.2.-J.4.-6.
7.9.11.12.14.18,19.22.
W~ ha~. in a prnious study.deftRed
~ 1.0 Kb
4 O., Kb
the 19aons 01the KEL F8C 01 . pedO8with coaIIDO8
Kell phenoIype.buathe moIecu.. ... for the K I JeDOIYP8
kaowa.'. PalyIlllX'plllc:difTcrcncesilie clUIOdby
WIll IlOl
... 1.
,...
-...- 11PCII
-
PCIIJ "
.
L.""
III
n.PCII~-l.1II
... IM ... ,
, 111M
IIIPCIIJ
'."
JA
-
...
di
8--*., ...
... ... ,
~
,
""
I'I"'ll"~, . ......
.. 1."
penonI of known K 1/K2 phenoIype.Included la
thae S81ftplaWCIeDNA from 1 HuUerifa, bec:... funiJy
!ItUCJIeI
indic8ld Ihdr KelI JCIIOIYpn.An od8erl8IIIpIa
were fJ'OlDwuel8ledperIOM. The 140-bp PCRproductdI8
~
eKOIII5 and6 (flCR J. Table I) W8I lreatedwlm 8SIf8
I. A KI/KI JC!IOIypedIouJd yleld !wo f...ameru. of S4O
and 200 bp; the UIIO pnocype will field the UIII:UI740bp PCR prodUd W1dKina hctcrozyptca should yjeld 3
ff88IRCIIU.",<40. SolO.und 200 bp.
or the 42 DNA sampla I"'"
12 WCIeeicher Kl or D
homozyJOla. 6 WCI1I
K I/D hctcrozypet. and 24 wae Kl. Kl phenolypa bal conlaåJledlow-prcvaJcnce
or l'8ft Kell
phenoIYJICILTheIe pllenotypell inc:ludedV. K6. K 10. Ko
(...",), . KI4IK24 he8eluzy.,.. and. Mc:Lcod~
c.c..
the /km
I FJ1OCYpina
..-
wim the
KeJJ pheftocypeslh8I Wenl deaermlned by !!eI'Olop: aulytris
o( RBeL In 2 ~.Ionotypinl
identilIedoneor ilie SImples
as K21K21111d
theod1eraa. Kl/Kl hcteI'oZYpll and theIe
2 'UU1IpIeI
were !CI'OIop.-aJlyidmtilied as bavinl "weu"
K I phenoIypel.
Nonc allhe otherlow-Pftvl1lenccor me Kel' pbcnocypa
Jisfcd8bovchId the C lo T buc subelitUli08ia C"GII6indicatinl fl... this c.:h8npil speciftc:ror the KI/K2 poIymorphism.&amplesof relec&edreMI'b lire 5bow1lia Fil 3. II
some CIIICI(lana 3. 4. lIIId 17). the K I homozyJ*l. in
addltiOlt ID d1owin8b8ndI ... S4Oand 200 bp. '-'Oftf8ined
.
rainl h8ndid 7-80 bp caUllCdby inawnpietedi~
The
..
rearr8I"""
M! daiped . !ICria o( pril8ell I" wouJd.lIplUy ... 19
C:uu
d Ihe DNA ~
of KIIKI
I1IIcIIC2IIC2
DNA. The only buc d88nte
dill would ax:ode
a different _no acid W8I found in aM 6 ud cb8apet .
lhnoniIIe 10 . medlioaiaeIII . conaenna N-")'COIYI8IIo8
mod' (As8.X. nr ~ Mec).T1dac:hu.. wouJdpI8Ve8tNo
linked ,aya.ylarioa .. Chi. s.18~.Baed on the amlaoIdel
~
of KeY proMin 01comrnoaphenocype.theft .. 6
paaible N-JIyaJ8yl8lioasi_lit 8IIp8nIine raidua 9J. I I'.
e:lOftl of KEL lIId
III
diffcftnl
In 40 of Ilie 42
maJ be c:IUI8d by
or ahcra8d~ aplkfn8.
To deIami8e the moIecuJao
buis 01 K 1lIC poIyll'lOtplaism.
--
~"'IIII.~"
.I lN4.. T'" ,TII8"""
.~
la lllllmo acicI
poildJon8.
191.34'.6'l7.-
n4. Howew:r.Ihe
paaiIioa
724 il prob8bIy noI pycosyl8led McaUM it ja p8I of .
se.-.
AlA. Pm. Ser lad tbe pre1C18C8
01proIiM bIIwea
... Jefil,1eItheo8188
In1Ubic8N-8IycoqIadaa.
.
01
dnaIi..
10 raedriordn8
..
po8Jdo8 193 would
pmJlycu8ylarionII
Ii- 191. Thus. Kl puein
would be compoeed014 illsad 01, carboIJydn8e
moiedea.
Anumilll u avaap ~
sia 01 .. N-lInked oIip-=clwtde 10be abouI 2.' ltD. the K I pnMei8 sbouId be 9CU
kO . ..ooIp8Ied wiU8 the 9J-kO proIeia tII8I c8ria the
CCJl1U8l8
KcII plKnotypa. " 1'hia dåft'cnnc:c i8 sim il diJIkuJc
to dI:8Ict bJsodium dodecyJsuJfaee-polya-ryl8mide pi declmphoIaiI
becaaMe
aJycopmccitendto mi
. t. --IIr'
,..,
"_'--L__-
,. na MI m MI - MI C18
.. CYT
ni .N liIr
a
-- -- n- -- - -...I. Ct8, .uti111 -.
,.
-
. diI1'uIe
lt1f
-
,.
-
na
-
MI
-
m
-
MI
.
1
-
.-
'M
- -. - - -. .
t8 ........
C18
-
CYT
nw
...
,.,
Pall ... 1 ... ,
" ...
. ... .
MIt. lin'" ... - -- we.. 11I~.M"
II DM. fte...
111 l T el .
el . ... ... ...
... ...
~
..
Ylleel II 118M
la - ... .., ... In
118M
- ... --la
C.. , IDI..1III
1fMla
kilt""""" . _. *'
laIn .. -Å-M
la,
.......
."""'ttlll.. la
"8
l 2 3 4 S 6 7 8 9 10 11 12 13 14 IS 16 17 18 1920
: ~j8 tg
.200 bp
... .. ..,
Kelpltl~a1VPne
filfet-- a ...A~"-'"
...,..-
...~..
... .,
JCHI.
...
~
11.11 , afal'aJflJ
-Ål.""""""'"
.....
t
,-
a... J"...
11. PCM'" Kt t Inl) .1. In. ~
J"""
tt
L~
J
-.,
hl ) .r'1
II tCI""<t ~""
..-,Ktllll""'-.~_""
II. _IL--t""-I
11.,
11«'" ...
tC:1,J;...". fL--1..u,.-4;'" 1&tC:-t.a.t.-7:'" ta
MotC:-,.a.t8:...,.. !C:-t,z,tfoMl""" 111'""'11.1L1.-1:"11..-,.. tC:-t.J,Al a
--'-"""""K111Ct
'--4>'" tetllllllWl".,~"I"'"
J
ilie-- '
-- ...
...
........
handa. NeYertbelna. unalyllia by WeI8em illlnlllJlObtolaIlSini rabbi. lIIIIibDdy to . syDlhelic peptide derivcd from the
cylOplMmicdotmi. o( ICeIlprotein.1,1 I~
!bli K I proletn mi
sHpdy lIhe8d01 the KeD pr'DICindill c:arria
comIIIO8 Kel phenotypes(datanot showa).Lack ola c~
,'i,,~
\~
hydn8e s~ c:haia ma)' CIIIX* differenl JWU 01 the proteiD
ladini 10 inI~tJ.
Thia ia 8JIIw oampteolof aJ)'COIYIIIion in l1li RBC surface pro8ein
10 .
chM.. in blood poup phenotyp8. The Webb aI)c0ph0ri8
C variaal allO lucb 811N-sJycllll..n
'The paj..
imI from C to T iII Om! 6 cra8a a new
mltric:tioft cnzy.. site, "-OAAIOCT-J'.
dI8I caD be cul by
BlM I. ThU l'CI8ricUoeCIIZ}'IIW
dI-allowIthediffenmti..ioa 01 KIIKI and K21K2 homozyJC*8 and o( Kina
hetcmzyJOfa l81dshuuld be I8CfUl II 18diapollic JCDOIypo
proa:dure. A pouible drawbKk II ch8I. in very rare cllCl.
RBCI do DOIclIJftSI any Koll and
In 11111
phenoIype.
knowlI - KolnaR).lite
R~
lIfIPC8I'
noc lo -..
..y KcU
proIeln (1ftthe RBC membnae. Yd,lftliminllry
capcrilllCllla
in our labcnIory indkalc dIIII 2 Ko penoaI C'OIM8in lCeU
mRNA in peripMraJ bloocl and th8a the sequeace 01 the
mRNA (101ftthe initiation A TO codon 10 the poIy A taill.
IcJendaJ 10mRNA abc.ned (rom penonI wldl commoa lCeU
phenoIype.mi IIndJn8Indicllles dill the ... sequenc:a in
lhe 19 CUIIII 01 K2 and of some Ko penOIIS 1ft Idcndcal.
Thcrcbe. PCR ampliflcatioao( c... 6 and pnoI)'pinl by
tIaImaII widl S. I wouldindic:8re
a 1(2 JC1IOI)'ptla Ko
pcnonL Scrolop _,sil
cull)' ddcctsthe Ko homozygote. buI tbe Ko bctcrozyP*5 would be ~
icIeaIilied DI K2 or K 1 and BVIt IlIIIalysia would allO nocdilc:riminate. Prrsulll8bl)' the gme would oa:ur i. the Kmod
phe8otypeI in wtlich cxprasi08 01 all KeJl-rebuaduri...
1ft waItaIed.
The PCR method dncribed could euily be appIied 10
DNA 1IUIIp- obWnedfrom amniodcfet&!celllllllll"'ld
prove
111
detenaiaiftt the K I ... K2 FIIO8ypa of the
fdUa.nål ... C8II pocea6aIJyIdcntif) thmc preananciCI
lit
risk rar hcnIoIytic:cIiseue 01 the newborn.
AEF!IENCES
I. ~
WL: fjllDDd
af -IM C8UII, iii
SwWw 5N (edI~ CliabI Pr8cticeuI BIoa6T,..,
Yart. HY. Cllun:hil-Uvlnpt-..
2. MIna!WL ~
bk1od.."., "y*-
I 911. P 79
CM:RecdI..elopmmllla . KlI
T-'UI
J. MInI! WL ~
review. Tr"-""
LO,
New
Med
1:4. 1911
CM: 1'118K.eJJbIaod lfOIIP s}'18a8:A
:JO:"..
1990
4. RNIrIMCM. M8I'IIIWL: 1111K.elI
and Mcu. ...
cet...'" All" PC,C
JPtå): l'IoeIta81ood
0.0.. A
01
ne ,...
bl Cell:SIrwIuIIr, fu8cdaa A8d CIWcaI Slpifte8ce.
~
Mo.
~.
Haf*i8, 1992.p')
CM, M8nIIIWL: 1'118
W bIood~
1'- MIl
tIIIeMd..eodpbe8alype.SeIIIin HemIIoI »'209. 199)
6. ,.., AC. DI8WI OL npp811P: Appllc8lo8 01 ... MAIEA
uaay lO. Ken bIood PvuP I""'"
Voa s.., 66:216.
I,..
7. ZeI8Iti T. CopIM O. Myll Y. 511.RIC. ...1..
..... --'- u. ,,
... ..,-..illducible
.
o
u
"'-
""--
"--'1
,
5, WhiII
- ---."--
pnIICillod: ProwlsiunII_JIIIIIeIIIol KEL
-
. u .-- .-.
101:_I. lUI8 n88 ~
:1:1:1.".1,",1
I. ~
MT. ManiIa8 N. MillI JS. "'RH. Sp8II' NK.
8D1d
E:
~
of ilie KenbJood
paap
locuI cKEL11ocInnIu8ome 7qJJ-q3j by nuurc.:ac8 .. lIitIIltJ.
bridlzadaa:Evldna fe.-dI8 poIypqMidt lIIIhII'e
of
v..
110M.
..
GenIt91:~. 1993
.,.
LlI ET Al
9.... SoZ8mII8 B. M8nh \VI., RedIu8
CM: 1118humu Kel!
.. .Jw8oIonI87qJ3... I.
bIoodpuup
i8 re-
lCrict8d10~
ccl'" BIaod 1':2804, 199]
lo. LIe So z.BD,
WL
CM: MoIIcuIIr
clallåll
pr!.-,
~ K8II bIDad poap pnI8IiaL Proc
N811AcM Sel USA 11:6353, 1991
II.
CM.... S.T-
- eL.
ay. .. MInIaWL: ~
lfaåIi8D.
~
139:-I-
BI.J..
ud c:hi1llpllll8l
lC8I bload P'DIIf ~
1\""'"
11.
Do MIIII8dI H. S1 Slak
A. ... D:DI8uIIIde
baadI
~
~ ltaI
CIftwrt8I8(Yl") bJaad paIIp
.. .
-..
H-
...
iDtlpity.SrJ
S4:S73,
1913
I J. R- D. LIe S,
W.
bIood 11'0IIII
ud die Ix"",-
C: TIJIIDIaD01 tbe Id
~ muJdpla
by
lI'IIIIfec:t8dcetla.BIaad 84:3-'11, 1994
14.LleS,Z8II8B.8eda8C:Or.. """"01.........
iq
KeD"'"
die
15.Jaha
SWM,
W
.~
cIur8 for
R8 19:401,1991
pIIIp"'"
o.
JIIIII8Ik
DNA f'nn
16. ZIIII8i T: 11Ie18~
BIaad 15:1364.1995
RoSc:rtwr
C'I.:AnpId..a.Ir.ocytIL NIId8ic AådI
~ DNA IWIIokåoft
~
wiIII bIDod
,.
paey.
~
31:762. 1991
17. Mu"
T, PritIcIIi EP, ~
J: MoIIcu&8rCbinF A
"""""'.1
CoId s,ri8a Harbar. NY. Cold SfIrIJIIHIIIIar
L.Ibu.~,
1912
II. OIbId Ea: A c:rttIq..0I1heIbeo
huInI o( n. VI
i:ill
ll8IIWioIL T,..,
1:233,1961
19. MlynlKM, Baw8' PI, PnII&
Ok".
lipilcu&:8 01...
K8IJTT ,'"-"iii pNJII8IIJ.ClillLIII H
II2:379,
1990
20.DllpIdJD(, lInIIIIiIDw
IM:~7.
ben.. ID...1(. Voas-. ":8,
,.
01dia...
p, J8IU8-DupIa
p, DailIo8Mo VI"'"
21. ~II
bI8a& V: A
cI-
1990
c:I8
01
dl.-
011118
Mo
o.-
newbar8
-- - ID
_-K (Cd8ll). Acta""""'85:45,
1991
22. C-"
"I.. O. PIIqjIIIIa8N, W
J8: And-1Cat11n
.....
.-y. 8r J ~
O~
91:943.1991
23.
HM. Oib8l1M. 8ImI8 sa..R8d MM: AJlå-K8I..
JII'8PI8CY.
ar J 0bI8II ay...I9I:I62,
1991
24. lIcnmIa 1M.Pallock1M.MInniD8f A.
ex. MnIiCUl- S:
KelI biocidpoIIIIlIIaI_0tII8II0,-79:239,1992
25.... B:SInII:bnI
B*,-
1 2O9:JJ
I. 19IJ
01N-tlycalyl............
26. Te118MI. Le V. Kl. C. 0IIiuD MI., CannI8 lP, CoIII Y:
Erydncy.. WIbHJpe "~Ia
C vlrillltl8cU N-aIJcoIylldae
due10..
801818 .1bo'1
A. l
37:",
1'11'1
27. C'IIu8S.RetdMoCoIIIIa7
J. K8nY. Moh8adM
N: MoIeC1IIIr
c:1wa:IertzadaJ
al erydInIcJII 8IJ~
In C v8ri
BIoad
17:644.1991
7)
IIIUIOII11T010&1
MåANITAL
Differentiatlonof autologousABa,RHD,RllCB,lUlL,JlC,andFl'
blood group genotypesby analysisof perlpheral blood samplesof
patients who haverecently receivedmultiple transfusions
P. Roim8n, T. Dovt, and C. Gassner
.
.. .....
MC:KGIUJND:
1*1 nUIIpIe
logIc ~
al
---
**V'"
II bkIod group phellCIIWI88
almbIIdR8Cpapll8loo8.
The
«9)
..lAt
- 8U8DIogoIII
bIoodgroup...
g8ft....
p8I8nI8enddanenW8r'8818IyZ8db8b8 end
~
8\I8få. An 8Wf8II8al U ,..
.
fram
21
-..
WBC-r8cb:8d R8C unII8 ... ... 8\8'8g8
888 al U
~
lIdrnInIII8r8d JI8r 118n8fu8lan 8'tI8rII.The
lr8n8bIan8
~ Al
.,. .. ~
AllO,Rh,K8I.l<Idd,
Ini ~
~
paeIIrwIIfuIIan
... bucc8I
5.3
phelåyp8db
framP8I8r* W8r'8
.. con8IPOIldIng~ . unIIIrm
PCR88qU8I
~
pm. JJR*IcaIIh8t
8IIDw8d
g811G1yped
b
lh8Ir8InUI8n8ouId8I8m118
.-do.....
3 hour8.
RUUL1'8: Al poIIr8n8IuaIan
lIIhIIII8d~
caI popNh18 al V8tou8bIoodgroupsy8I8mIan .....
logIcl8IIIng.Gel~
fram...lpheIaI bIood~
duc8dl8UII8 Id8nIIc8IID
.. . ~
II bIoodgroup
phallCIIWI88o
I~"'"
al.. amount
albIood...
fuMd 01al "18ngIh al..
ru.Ion.
l8IIpIng p8fIod8ft8rnr.
CONCLUIION: A f88t end raII8bI8
åftc
tI'U1IIuston
ABO/Rb-comp8dble
pr8dk:e
requJns
RBC
units
ue
th8t
trula-
fu.secI to . patient. Other Rh systemand.....
such. c. c. Boand e, . weil. an,.
In the
KeD.KIdd, Duft'y.and many otIw systems. ue not maldled
unIeIa the p8dent hu deYeIoped respectM aDoandb0cUe8
antIbodIes can C8UI8hemoJystI of tr'8nIIu8I
RBCs.dIII'8r-
endadonof ortpI8I bIoodJI'OUP
an..
lt8tUI II lD8Odatory ror c:onftrmadonof andbodyldetadfbdon and ftutIwr
..
traDIIusaon8of aIIIIp8db1,e RBC unttIln such cues.'
Aa. . pedent
rec:etved
mu1dpIetraI1IftJ8Iol18,the
donorRBCapenIIt In bli dn:uI8don ror weeb. and thecWrerendlldonof ortpJaI bJoodPOUPphenotypeIbecomes
difIcuIt beca8e~ themI8d-ceI P'1P"""'" ~.
espedaDy"no pretI'8nIfu8Ionspedmen II 8Vd8b1e.1-3AJthou&h
the padent'aown RBCacan be l8p81"iIåIdhm the transrusedor8 by vartouamethodsand typedserolop:alJy.JIn
pmctIce.the l8IUJtsarenot aIways~~
n.nbe,
DNA medIodI o«er aIc8madve me8JII 01decInnInIn8
artaIn8I
AllO,Rh.Kell,1Jdd.~
andod8 dInIcdy Important bIoodpoupa. The PCRwith sequence-sped8cprimln8 (PCR-SSP)
to be moIIt U8Ad ror this purpoI8."lI
AIIbau8h
pnedc typIn&can be pedormed on dIIue8
such II naJI8or bw:ca1 ceUa reprdJesaor tranIIusIon or
-
--
PCR~.........
pfInw
DNA
glllOtypk1g
~ b
alauIDIogatll
AllO,
blood
onIy
due to prevIou8tr8nIIusAonsor prean8Ddea.
I Becau8e
RBC
STUIWDEIIONANDl8THOD8;BIood
...
pertodal bIood""'111
umnt
18
..
gellCItW*1G
al NJA Rh. K8I. l<Idd,endDuIfy....
CDIMbe \.-lID ~
C
.
Rh, K8I. KIdd, end ~
bIDodPJP8 canbe ,:J8fbmedan ,."",..,
lfD9I thepaIenI8
t-.. ~
C8Md muIIIpIe lr88fu8IcIna.
bIood
,.
_~I
~
re8trtcttan
cilie
~w
P"'P"-'-'W;
.
MU".
1eqU8llC8-spe-
~
Fromdie~
Ii al ~.
BIood1'r8nI6asIonC
al SIonnI8.1Jub
sau-.18:endtheC8IIIr8IInsdtuI8AIrlIIDodTr8nIIuIIonendlJnmunoIuttc8I
~ tu-1I,
Gerw8IendUMwIIty HOIpbI01II1I1Ibnd.lnnIbruå. A...
trta.
Adrt88,."nnt ,..,... ID:PrImoi Roim8n. MD. Dep8tal ~.
BIood1'r8nII8on Cenå'8al
s
~
I. 51.1000
ijubI8n&
SIowwII8:..111811:
p~unI-'Å
AIr1""""'-'" ~..
14.1_1ftIIIoa...
ceIYed
Ju.-y
II. 2000.lIId lIX8pI8d 18I1U8Y31. 2000.
TUNSftJSION
131 TRAN8flU8IOII1VolWM 40, AuguIt 2000
. ~
tqment JenadIpoIymorpIdIm; SSP
2000:4O:t3I-NZ.
GINOTVPtNG N'TIIt
transpi8nt8tJonhlstor...1Z pertphera1blood samplesare
more aslly obtaJned.R8c:enc
reporUIndudeclravonbl.
~D~~~~~~m~~~
but the technique8UIeddId not aDowlmrned!8teacquJ81don 01resuJtalJor Wen!UmlhId to . determJnadon01Rh
polymorph8m. 14lnthis study.we thenlore testedthe 8bIIIty 01. nnIy "-4~
uniform PeR-SSPprotocoI ror simultaneouaand... det8mdn8donoI8UtoJoFusABC.Rh.
J<eJl.
lUdd,and ~
mttaen au m the pertpheral bIood
samples01p8denb who repeacedJy
recelvedmultiple tI'an8fusionI 01blood mBtchedfor only the ABCand D Indiens.
PretranstusIonand poettnnsluston samples from etaht
patients andthe correspondIJ1IBBCunIt8 thai weretru8l'u8edwenI both pt.notyped by routIfte seroJop: testsand
genotyped.The resuIII WIn then comp8l'l!dto the reaa1ts'
01bucca1DNAtyp~ 01the patients.
MATERIALS AND METHODl
AllO. Rh. KeD.XJdd. and Dutry bJood POUpl Wen! Jmesd.
gated metaht
&duk p8dent8 who have repelltedIy receIved
MULTIlU T'MN1llU1ION1
tranIfusIons. AD~
muldple Ir8nIfusIonL
penonI (3 men 8nd
5 women) were whJte and h8d or bad expertenced
CI'8rdoph8rtnaI
(PIdeJIt 1), po1ytram1a with
withrupcwed
spleen (PatIent 2), myeloftbrosta (Padent 3), poJytrauma
polytrawna
(PIdent8 4 and 8),l'nIctured femW' (PatIent 5), coron.,
surJ8J (PIdent 8), end c:ornpbdanI8ftIr
ren8I tr8nIp1antatJon (Patient 7). Patients 1 throuah 8 receMd multiple
b'8rIIIuIIonI al p8Cked RBC unb on 3. Z, 2, 3. I, Z. 7, and 8
0CC8II0nI, rapectMIy, tbr . tot8I al 28 tr8nIIu8kJn ewntL
The tI'8nIbed RBCawerenot WBC reduc:edbelcn tnuwfusion.UnIII 01RBCawer8 _Jected
to match p8dent ABO
~--_nandaen
st8tUL
and
D
lIJd&en
statua.
TheIe
un1ts
were
befon dw tnnIIu8Ion lMInL PM p8d8ntI ~
fOR
the
tranIIu8ion
nent.
PM
p8derds
(PIs.
1,2.4,5,
and
7)receMdlDIn8 ABO-noeddeaö:.l RBC
ABC-r1OI
noIddlaldc..I
7) nc:eMd lOIJ18ABCddendcaI RBCa
RBCa(aDbIood poup
O) bec8use of the !C8I'Ctty of blood of Id
_nn-
~~oI~~albbd~~~~M
awnp
018.1 non-WBC.reduced RBCs
W88
d per tr8nIfusIon ewnt (r8na8.1-381unlts)
tr'8IIIIu*m
ewnt.
8dm1ntsteredp8'W88
the tI'8IIII\II8d RBCa
5.9
(ran&8,
- - days
.
- --""""""""'"
the b'8IIIft8ed
RBC8 ..nmltn..~tno'
erqe perlod oIbIood
...
-_o.
5.3d8J1f
..
__00
VoUn840. AuguIt
2000
TJtANIIrUIllON
l'cP-AJlI1' AL
The perlph8'aI bIood samples 01the p8denC8
cdlec:ted Inta lt8I1dIudEDTA tubes beIor8 the ftnt tr8nIAIaIon
ewnt. - weil. ... e8ChtransIuIIonewnt.Thebucc8I
sampletwer8coDectedfrom mouth scrapeI8ft« eech
plldent1l\8lt tr8n8fuIkm. ucept ror PIdents 1.5. and 8. who
had dled or had been moved to another ha8p1taL The
samplel 01 don8ted unJC8were töen from the standard
pilot tube8 with CPD.
The ABO. Rh. KeD. KIdeI. and Duft'y status of the
pmnuuIusIon and posttru18IuskJn gmpJes from the p8denC8 ... deUrmIned MI'OJoIkaDy and by DNA typln8
methodl. ...
each tI'1InIIuIion event. The pIIenotypes of
l:r'8I1Sfu8I uniC8wen c:W8.1I1h.edMI'OIop:aIly. AddItIon81
pllOtypb. 01 the buccaI sam'" 01the mdI8bIe padenC8
(PatIenCs2. 3. 4. 7. lind 8)
pedormed. The l8IUIts oIPCRSSPpnotypInc for ABO. RHD. RHCB. KEL.}1C.and FY lod
01 the pertpl8al bIood sunples drawn betan and after
eech tr'8nIfuIian IMInt were then compered to the IeJOIop:
...
rnub
81111to the nsuIts
01buccaIplOtJpinl.
S8roIogIcbIood group typIng
The ABO. Rh. KeD.KJdd. and Duft'y blood group phenotypes
were determ1ned by usInR . commerdalJy
avallable881card tatIn8 system (DIaMed-ID MIcrotypInl System.
DtaMed. Cresaler. Swttzerland) and commerdaI blood
grou~typIn& re..,.ts (Biotest AG. DreIek:h. Cennany) Kcordlni ID the JI181UIKturer's Instrucdons.
DNA blood group typIng: 18Oi8IIon 01 genomlc DNA
hrtphenl blood sampIes 01donan end n!dplena were coIIeded kl J!D'ä Bua:8I cell tom moudIlCI'IIp8 d lh8 pIderD
'MInt ~18d
In O.9-.-ant N8CI. W8Ihed wtIh PBS(pH 7.27.4). end .-.pended
In I mL ofPBS. DNAW8S--.el from
200
el wt1OIeblood 01' buCCII ceD lU8p8IISIon by usIn8.
blood kJI (QJAamp.QIAoGENCmbH, tfII88n. Germm1y) Kc:ordIn8 to the marU8cturw's bIood and body ftWd spn protocoL DNA wu qllU'lt:1fted by uslnl . spectrophotometer
(CeneQ\8nt U. Pharmada BIotech. UppuJa. SWeden).
131
TRAN8IIUIION
VcUn8.eo. AuguIt 2000
Ge«mY
..
( tV:;;
If!? \
:
ror the detectkm
T1W8II'U8ON8
SWtt28rI8nd). Concentnldons
w.
IW. KldII, 8IId Dutry genotwpIng
The aDeIIcvariants 0I8lJ KEL. IX. and FY
A
gene lod ... clearly disdnpJsbable by
spec:Iftc point mutadon8. whkh aUow8
the deCecdonola sIntIespedftc nucJeotlde !8qU8IC8 dtfla e.1Cebetween KELl
or KELZ.)1CA
01' )XB. and FYA 01' FYB. WIll
performed the pnotypinI 01KeD. KJdcL
and Dutry SJS8mS
by a newIy desIIP
aed,
unilonn PCR-SSP protoco1. uslnl six
penDel PCR-SSPL
The sequences01prtmen detecdn8
the K1JL.]lC.and FY l8f'8 lod we... desllfUld to lit the l81li8 PCR C}dIna ....
~
MUl T1PLI
01 pm.. for the detec:don
01 RHC.DL. J/C. and FY Wf1l8 0.25 JIM 8nd thoIe 01the contIOl prtmers1Nft 0.01 JIMfor 1CBL. J/C. and FYand 0.01pM
for the RHC.-pedtk re8Cdon.AmpWlc8tJan
carrted out
po8idon 8. whereal RHC-endIor RHc(qt4I)-1pId8c reKtJon In aon 1wu movedto ractJon poIIdon 13 ur-. 1B).
men
. was
used
N"T!R
of
BlId RHD. I' Prtmer sequenc:es ror
pnotypinl the lUlL
Iocus with the
aDeles. c:odIntfor the lik polymorphlsm. were choIen on the bals 01 the
prevtoul data.ll.l'n.
same approach
wu taken In choolln&the prbnen for
FIOtJPInI of the ]X pneaoa.. with the
common aDel..)1CAand}IUJ. codlnl ror
the Jk8/~ po1ymorphlsm.ll.l1.1I. weil
88 the prtaBs ror pnotndnl
01FY....
loa.8 with the aDeIe8FYA and FYB. codini ror the Py-~
polymorphtsm.1."D
The DNA lIIqU8IIC8I 01the prtmen U88d
f 01' KeD.KJdd.and Dutry detIecdon. theIr
names.IDCI theIr comblnatlon8 In the
varIou8 l'I8CtIon8 1ft! 8fW!nIn 1åb1e2In uch of the sbl para1Iel PCR-SSI'I,
a 434-bpPCR hllJD8l1t from the human
growth honnone Iocus pmltlon 5559 to
5982 W8I c:oempJtfted. a ~
contro1~
to an 8It8b1lhed method. D
8
.-qv------.
1 2 J . . . 1.
. t. 111211 R-'Ian
R-*'n
1 2 I . I .
numbIr
numbIr
ConInII b8nd
(434 bp)
ii~.~~
! i! g . I I '!
il! I I !!
SC*IID
I I 5 i
PftICW(bp)
. J
Pd8nt 8 8fIIr nn.fU8Ion
I.""'"
-"\'",
..
...
II
"" fII",-",= ~...
~n.. L . ~+aIIIt,
C8å Pr.- Cb8..J
J... """"""""al
ta
.. ,."
.. .....
~~...
.-;t)'"
fIICb8IJ CIIII888,It.............
- C+c+~;K-II+:""");... "
b~...
~
"'*il ...
..)..
Ev8lu8don 01 the ~
PCRthIt
,
-"""""""" il"""""""
SSP genotypInB technlque W8I per- :.twd D DC"""
Mt7I 111
,
il
fonned by tBdn& 300 DNA umpof
c.D,.. 1.1Jt'.
~. ".. "-,,.. A8
known KeD.lUdd. IDCI~
phenotype.
III"'
~.
il
ForKeD... lUdd,aDaenotYPInIleSUJts
",,1Ir
1IIID-9III
3,
4.
s...
7.
..
...
II
correlat8d predlely with the known selid'
.
~
J
..
JII8I8hoI;Cb8,...
rotype8 (data not shown). SJmtlarIy.
ev8luadon 01DuJfy pnotyplnB rna1ed
..-III
JrIUDU""
~
IDdie A...IlS approp1al8 8CCUr8CJ".
further detadl
8D....
\;
pIIIIIIIIL
AcanI8
III'"
are de8crIbed eJInbere. D
fil...
~.,
... MCPIIII
ca.- C.
.'
,...
..
-oM.
-
.
~...
~...
PCR pm. seIecdDn- pedormed
with a computer pvpam (M8cVec:tor.
venlon 405.3.Kod. New Hawn. CT);
-
oJJsonudeoddes
synthedzed by a
synthesll service (MJcrosynth. BaJpch.
..
,...
"",~ Jo ""
. -...,
(Hal.z.tt.
c88llll1IJ".
D+_.1"
"-'I'
...
(lit.. ... ...
.....
.~.
-
L
-
~~.,.
~.........
_.~
..
..
...-
.
".
-:J MIlt
lir. ..."".
.. . dlllIID
(NIt..'). ..,....
II"'" alb . alKa.
~
MIlt
.. ... ,
... ..., .. ... t'.II8dJ.... - a. a
...
"InII1....
........
VaUn8 40, Augu112000
TIWf8PUIION
131
ROiMMITAL
In 8 final volume ~ I O.-Locontatntna50 mM XCJ.I OmM
Tris/HeJ(pH 8.3).O.OI-percent
.latln. 5.0-percent
1IiJ'ceroI. I 00 ... per ml r:I a'88OIred. 200
...vr:I e8dJ dN'I1'. I 00
mM W88
second8.
94"'C. 1OIncub8donc:ycleIfor I O.xnII8I
g.rc
and 80 sec:ondlId 85"C. and 20 Incub8tlon c:yclesror 30
second8.H'C. 80secondIIlIt81"C.... 3O.xnllu
72"C.
PCRhtPents were!ep88ted by sI:IeIn . 2-percentapr088 pi contatnin8 0.5PI per mL or ethkUumbrand" vi~..I""'"
with uv 1I&ht. and documenJedby ~PIi.
RESULTS
1b detemdne the Ippralllm8te l8I1dtvtty ror contammatian with roretanDNA of our PCR-SSPsystem. one represent8ÖV8
rncdon (AB() reectIon 2. spedftI: ror non-Ol se~
.. tIII88d
withmI8d[lilA58IIIpIeI
~ heCI!rozJpIS AIOl and homozysous 0101 DNA. and the mtnIm81
absolute unount al AIOl DNA needed for a reliable PCRSSPstpI WIS establJshed..The rnJnImaI amount ~ AlOl
DNA needed ror a reliable PCR-SSPsipI8I W88reJadveIy 10
pen:ent and the rnInim8I abIoIuC8 amount 01 AlOl hetengyJINI
~~C
D N A needed ror a weå
TRANIPUIION
samples.wben t8Steet(5/8 padentl).
Theposttr8nafusIon..1OtypInBIeIUItIIwerenot 1nfJuencedby eMherthe unount orblood tr8nItuIed In a b8Wrustannent (rmp. 1-38urdtI) or the S8II'IpHna
periodart.. a transruaIonevent (1"8np.severa1hours-Z4 days).
Patient 4. ror 1nst8nC8.who la A; D-C+c:+E-e+:K-t+;
jk(a+b+):and Fy(a-b+),rec:eJved
Z 0+. 1 E+. and Z Fy(a+)
unJII 01 bIood dur1n8hJI sec:ondtramrusJonevent.
CetIOtypb. or the S8II'IpJe
th8t ... drawn lmmedIaceJJli-
t.. tr8nIftJSkm
l'IIVe8Jed no RHD. RHB. or FrA 1II1e1lcmat8riaL wh1n8 the seroIop: teItInt round MCPs In C. D. B.
Fy8, and FyllphenotypJnt. SImiIarIy. Patient 8. whma autolopaa phenotype la A: D"+C+c+E-e+: K-k+: ,Jk(a+b+):
Fy(a+b+), rec:eMd 4 unAtII(3 B+ 8nd 1 K+) ornon-WBC-rw-
duad RIICsdurtnthI8fint tran8fusIon8ftI1t. ~
undertaIcenon the sameday reveaJedno evldenceor donor RHl/and ICBLaIIe- In hå perlpheraJbIood sample.
SeroIap: typInB.howMw. rew8IecIMCPsIn pheno~
E. Ko.Jk'. Fy". and
ryaan...
(... 'Dible
1),
PCR product W88 l O 111-
In aU pretranaruslon samplel hm the padents. the
ABa. RHD. RHCB. D!L./IC. and FY l8ftOtYp8I and the all'responcllnt serokJ&Ic phenotype8 correJ8ted prIIdIeIJ Aa
expec:ted
bucc:aIDNA typeI te8ted (Pta. 2. 3. 4. 7. and 8)
were IdendcaJ to tho8e 01 the respectIYe stardnt blood
samp1e8. Resu.lts01 serolotJlc and DNA 1mestIpdon8 ....
pen In TIIbIe l.
It.. evident that aD 01 the 28 ~..'*'usIon
samplel
exhlbtted MCPs In varIou8 systema oIblood poup antf&enI
as seroIo8kc:onsequencea01muldple tranIlusionI al nonIdentk:8I RBC unlts (see'DIb1eI). AJthouahthe Intenllty 01
MCPs on pi cmla rouahIJ depended on the number 01
ABO. Rh. JCeI.lOdd. and ~
norddendcaI unJts that bad
been transIuIed per tranIIIusIon ewnt, ene can nate that
the transfusion of even a sin8Je nonJdentlcal RBC unit resulted In an MCP on sewnI occa8oJ.. Por lnItanat. P8dent
8. Wh088 autoJoaous phenotype II B: D-C+e+IH+; K-k+:
Jk(a-b+): Fy(a+b+). recetved 12 unlts dw1n8
his fint transruston. 0nIJI unM 8Il1O" theIe W8I 1+. wtddt resuIted In
an MCP In KeDphenotypIna efter tI'8nIfu8on. Alter the aecond trarIIft.8Ion ftent, when 2 0110 unIts were K+. MCPs
penIsted (see'JWJIe I and'"
lA).
In aDpetlents. the resuits 01ABO. RHD. RHCB.JCEl..JK.
and FY llllnotypIJ1I ~ the pertpheral blood samples aft8o
multiple nnsIusIons coJndded completely with the resuItI
NI
oIbocb serolotk pbenoCyplnl 8nd IIf'OtypIna befont mu1tiple Ir8ndJsIona (Me FIl- I 8), AD rauJtII or genotypIJII 01
pertpher8J
blood... 1dendca1 with those 01 buccaI
VoIIm8 40. AugUIt 2000
Ontheod8 hInI. lOI1Ie sensItM technIquescandeteet certaIndonor DNAlII8rken alter transIusJon.1\vore.
cent stucUes reported
.
-- --
the eDstenc:e 01 donor
DNA In the
perlp1881bkJodof recipientsup to 8 weeb alter tnmslu-
GDCmJIING
slon. with al appro8Ch t8ed
on very senUtJw nesud PCR
techrdqua...
The crltic8J apect al the rell8bllJty al methodI ror
blood IJOUPDNA typIna after multiple b'8nIIusIonIls the
methodl' abWty to deted Iow propordons of DNA In mbI8d
S8ITIpJeLHesIr8' et al. J1demonlCl'8hd thIIt the danar DNA
In the 58Jf1plefrom pertphenI blood C8II be suJIIdent ror
PCR-SSPaenotYPlnIwhen prnent In the ranp 010.2 to 8
pen:ent. A comparable PCR restt1cdon Irqment Ien&th
poIymorpIdsm (RFLP) auay h8d a much Ioww sensitMty
al only 13pen:ent. »The threshold lIJIIOUDt 01 foret&n
DNA
thaI can be shown by Dur reprl!M1'lt8dW lII8J wa 8bout
1O percent. whk:h makes thI8 a robust tesdnt system. lt 18
lJ\teI'I!IIq th8t thAI reIadw ln8ensIdYtty al DUr PCR-SSPI
seem8 to be of advUIt19 ror the pretented purpo-.
In OW'study. we attempted to seewhethlr OW'urdfonn
PCR-SSP protocol ror sImultaneous Pnotyptnl 01 ABQ
RHD. RHCB.KIIL. $. and FY an
whJchcan be perIanned la a
~
wIttdn 3 to 4 houri, W8I canY8I1Ient re. !UChdetennIn8don.ResuIta Indeed show th8t.
aft8I' muldple tranIIusJonI. DUr PCR-SSP...1CItypIns fmm
perIphenI blood samplel reveeIed no ott. DNA t:ype8than
tho8e oI8rwd In pretrarIIIlusbI sampIea.whAchaDowI the
condullan th8 onJy autolcJtouaDNA wa demonttrable.
Given th8t al approprtate interpretation 01pnotypåIR results Is uaed. the IftUJIS seem to be conslltent. fI!88I'dJe8
01 the qUUldty 01 the transIused blood unltI. theIr ap. or
the pertad aft8r wbAch the sampJes wenI drawn after the
tnn8fuIIDn
evem.
The reIUIIs
W8I'8 not a6ded
fRAN8IIU8ON8
01p8denII wtth c}toperU; In suchc-.
mG8tprob8blyshouId be UMCI.
buccall8II1p1e1
ACKNCMlIDGII8fR
,
n. 8Uthan
Ida
dw U88IIaI8'
n., tII8nk~
-
1klor..01
SChO8111a..
MO...
heIptuI dt8c:uuW1I8IId for c6rq ~
on DNAtypq 01
bIDodpIpI. lIId ,.,. W
MO.lIId WA f1I8ILMC. ror
An8IDAt LubInIIo, PhD.
---
hefpluI""'"
wIIb nIp8d
ID RHC deI8ctIon.
REFERENCES
I. V
TyIIrV.ed.TecInc8IlMIIU81.
12thed.Bed-.d8:
Am8tc:8n
A80d8donallIIaod"""
11181.
2. Hudr8Ibeck
W.bnd S.SeroIap:8IIndIn. 8IIdelBctency
al DNApI'IIlIIn8 in trwIIIu8d
8nd th8Ir
an:e b ldIndtJ8ndpIDmItJ
~
....
Int J lA8II Med
191M; 101: I n-a.
3. IIIIU PD.AnII88 Dl. AppIIedbIood poup
,.
4th ed.
NC:Mar1pn8I
YSciInd8I:
PubUCIItIonI,
1-
4. Y-
P.aa- H.WhM8
T.et II. ~
!-il althe~
bIood..,
ABO
fII'IÖC
N8Ue
19110:345:221-31
5. O"'" MI..Ch88I8rMA. A r8pd UId åmpIe ABOpnoIyp8
. rIOV8IBIO2 YL A/02
JII8Ihod
1J1111II~
""""'-'
~,,"'It
cIIac:rtmbIat-
at the ABOIoaa VOl ~
1985;81".242-1.
PM.et ... RhII. ..1OtJI*II bJ
8. F- 8f. SbI8k S.
by the num-
ber 01 non-WBC-reduced unlts of blood mat were rnar.fused. HaweYer. the munber al potendaDy Incompadble
tran8lused RBC unItI sbouJd be conslderecl. and Interpnt8don 01~
resuI18shouJd be done with caution.
Al 18111801 DNA typb1&01peI1phen1 bIood comdded
with tho8e 01 the buct:aI samples. whkh aIJowedthe conclus1on tb81theIe methodl can be UIed for the determination 01 the autoJo&ousbIood poup pI1enotype status al
adults aller muJdple tranIfustorw.. ThIs Is al bnpnwement
(MIl' the 58'DIo8kmethod8.whkh showed MCPs.
Our study only evaluated 28 multiple tran8fusIons
amon. elpt patlen... Therefore. we c:annot exdude the
posaIblllty th8t rhere mJiht be cfJ8cord81tfIndJnp In same
paöenlS when tesC8dby feR after transI'uImn. The situation mlflht allo be cWferent In reprd to n1U8IYe trIInIIuslonl with bnmedt8te samplin& alter the admJnlstntlon al
non- WBC-reducedblood uni.. and especlalJyIn reprd to
1nIants. In whom donor DNA h. been detected by PCR24
houn
the tranItusIon alblood. JLDThenIore, we...
with Wenk UId ChIararI" that the transf\.mon UId traMplantadon htstorte8 remaIn etMntI8I to the InterpretBdon
01 pnetic reM" Ior 1nIan... patlenlS with bane marrow
tr8nIpIant8. and tmmunocompromII
padenta: In theIe
sjtu8donl, the raullI or DNA tyJJInI should be tnterpreted
with caution. Sped8I problems couId artse In the pnotypfnt
NIT!R IIUUIPL.8.
'"'~
1p8CMk:
prIIMr empllfIr8CInn. BIoad 1IIH;I5:82I-32.
1. MaIDJ8onG. SooIS, Sd1dTJ.et 8l Mut8donaIn the ..,..
rocyt8d8naIdnI rec:epIar(DuI'y) ...
ål alIh81'y81F)t1811d81na
8nd
~
the moIacuI8rbe01. d8Iedan
IntheDIdI'y
... elUI
Id)lII8IthJ
lndhtduaI wtdI
tII8 Fy(8-b-) phenotype.
J H8maIaIIII85:90:I23-I.
..
MI.McFuIandjG. I!ndaanDl. ~
el ICIlI.l
.
W bIoodpoup ~
UIdBU al thehl-.
poI)-I.
1'r8n8IUIIan 11188;31:485-1.
GMIIw C. Sd1manIa A. NU11b1wr8 W. Sch0nttz80 D. ABO
~
~
by~
dan wq ~1P8dIC
I O.
bythe
dI8In reKdon wttJaIIqII8IC»-Ip8d8c ptm8I.
c..w
...
C. Sch.U8/ da A.
by poIym11'88
chalnl'I8ClIIaod 1-':1152-1.
lIlJa-NOllar s.at ...
cll8ln n8dtan
-.
llHD'CB l7P-
MqU8I1C8-sped8c
prIawI. TI'8I18AJ8on
1991:31:1020-8.
Il. OU-.. MantmanM. BaIDy
P... 8l ThemoI8c:uIarball el
dielUddbIood..,
paIymorpI8m 8nd ... lad el -Hum Mai Genat
dIdon wtch typa l CÖIbet88...pdbIIty.
1911;8:1011-20.
I Z. MaJ'WI. GIock8. DNA~
el padentIwtthpotend8Iy
modI8ed
padlmllII pertph.. bIood~.
TnInIIuIbI
118;31:51
0-1.
13. 1WdMl,11IaaMoPuweI Y. a.laa-PIIrre D.BIoad pip
..1OtJPi. el bIoodsamplaahm ll8nIIU8adp8dInts (Ib1UIIct).Tr8IIIIuIIon19III:3I(SuppI):38S.
Vokme 40. AuguIt 2000 TRANIPUI8ON 141
RoiMAN lIT AL.
14. Lepr
TJ.1!b8r SW. LaIIoInek Klit'"
andllHa ~
... cornd
AppIIc8dan of RHD
bkIod
waup
~.._..
tian In d1rar*8Iy tr8nIIuI8dp8dInt8.TI'8IIIIuIIan
Z5. Wri RI!,CbI8IIrtFA. DNAt7JIInI
~ l'8CIp8ntbbId lftIr
lII8IItN trInIfuIIan. TrInIftaIIan 1I11;3'1:I1"'IG.
ZI. TInt A.MamIJ.StodIIrJW.1.euIIocyI8
""-111
m8I
trInIpI8nt8dan.
1911-,31:152.5.
15. K88p TJ,PauII8r MoCantU a A ...comI*1Idon hat spat In
the Ah
reve8IId by 8I181y8I801unnIM8d danarwwttb
the
r88
~
-~
pe. Am J HUlD GenIt 1_;51:
1018-73;
emåUD 8pp88ft Am J Hum GenIt 1917;10:741.
18. LeeS.Wu X. R8d K lit .. Mo1ecu18r ball of the W (K 1)
P!W1OthA BIocxII915:85:tlZ-I.
17. LeeS.N8Ime os. lWd ME. Redm8nCM. MoIec:uI8r buI8
forthehlp-Inddmce
18m. 1'r8nII'uIk8
II.
JI'CIUPI. 1'ranIftI8
b..1IpOrt
.
.1r
lInuI8
TPOX.niD l. D1S1O.HIA.DQAI.
bIood
...
dw CSF1PO,
n8II8d~
-
DB. II al The
01-""" Ip«dk poaym... chaIn~
conam 01DIII88m8Ieotlt8a818dDl.wNn
..
..
-
wIIh..
ptaI8'I (PCR-ssp):8 caudan8rJ
8Jaod
1991;82:"'8.
t.(f. AfaIIIIIA
A.
31.
C8ft
~
lt)
obriI8
!8npIII..
;t)ped AIr lmInun8 t}tope. IL cUIordIn. Am J 0bII8t
Cenamk8188;4:471-17.
l!nuI8
P. DNA typå1I and bIood tI'8rI8II8Ian. J PannIk
33.
GynecoIl987;178:327-33.
1M ni. DonI8InI. SIdår S. 8I.-:b Mr. Tr8nI6Int InCI88
In
~
"'18uIIoqt8
,.
IrInIftutcJnIIn bnmwlOCOlDpet8l1t
redp"..D compMIbII
wtth danar c8I proIIfIndan. 8Iood 11185;85:
1D- 14.
Wq-1IoctrIpa 1.Fry I. Mamno F. 8t al ~
01d0nor ~
foIowtn8l1'8n11u81nIn
(abIfna).
TIWIIIu8on
2000
In pertpberll
bIood trandJIIon. Sr J
dI8InnIIIICdan
~
~1PId8C
with Mu mI8Mr88point muIIIdon8 In la,..
and C8II be
d8I8d8d by PCR-SSP.
ImmUIIDI,
(In ...-.
23. ChenBY.LI8o YC.Smith DH. lit 81.1b8 human .-vwth horlIIDII8 1Dcu8:
~
btoqJ. and ftOIudan.
Set 1_;41:85-7.
pntI'8nIpI8nI8d
H--mlllII:l02:10CM-8.
BIood 1985;85:822-1.
TRANtPU- Volln8 <tO.~
.
LDLI. CYPA, HBGG. D7SI
AS,&ur- M.C8rundaID
L
D8t8cdanal
30.C8It8r
rnIcroc:Idn8I8n8ft8raIIap"* blaodtI'8nIfu8Aon
....
32.
la
P. ShpIb8n M. FnIUnII M. M8nny N. 1111eI'ed8 01
bIaod lr8nIIu8anI on PC1tDNA tJPåII
22. c.un. C,Kr88RL.Oo\'cT.et.. DUFFY
n« la---ted
24.
. In: Henry JB,Id.
CIInIcII dI8pIDIII""II8I~"
bJ""""'7
lIIIdtodL lIdI ed. PtdIIdIIIph8: SIundIn. 1-=514-111.
C.lA Y8I1KImC.c.n. P.lit ... MoIIad8rball
8dd re*Iu8.
II .
,. I~
el
C1In 1Ma11815;Z:3OI-15.
and PeR-DNAtypq 01the PyaIPybbIoocIwaup poIymarpI8a. Hum GenIt 11115;85:401-10.
21. ~
S.Ond T. ~ II. lMmocoS.GencIINc
~
tian 01the~0C8tn
D... DuIJY
bIood IJ'OUPPy"/~
a1Ia8ndpn.,
18-.-t
wtdI poIynD'phI8m
the "...mInD
..
lI1d CCIocLJ PCII'8I1ItI:Sel 1917:41:1154-1.
bo
'" pra88In.~
tir lpIIc8 ... muI8danIln
JbuIIIndIYkIu81I. J BIai Ch8D 1~13: 1Z813-1O.
20. T~
011
T~
21.'a.o
v nJOrdeIdoIlItsr.DouaI8kII.l8m.k",,_Mlm et'"
m
dIC8dIOII Of ~
In pII1pftefll
and lUdd bIood
II. LudIn N.SIdom-W8II8r
P.0IIv8 a... Cha..;tc.~
01 die
enc:ocIn&
the hunI8n lUdd bIoocIaroupl-
'(.
19811:1:424-1.
ZT. HuIdd8anIF. o..,
01theW bIoodpoup.,.
1917;31:1117-22.
C8tn1nJP. RIpacheP. U
IV. The e8Id 01bkJod b'IrIIft8IanI
kacyt8
typq by~,.
I99I:38(SuppO:38S. I]
1911:38CSuppG
:315.
~
~
~e'
ilC
',{
Q~.~:I
all E/e GeaotJpl.. bJ Allele-Sp,cl. Prl..er A_pllkatloa
By 8.H.W.F
S. SIm88. '.M.M."""',
M.A.M.av.bMII8, H.T!LM.~
A.E.G.ICr.
VGft dIm lIam8.
8nd c.J. 'ten dir SchoCII
. ..
... .. --
.. Al lJIU.,
tM lit CIII8
~~I'"
n. lit CIII8 ...
.. IIIQ,..lo...
~
flII.....III.
III. ...
.
Il 1:1
tfl
..w WII'
~
II
~
-
..
-,
III
..
... - .. ..II". ......
..
;IWI
ft8IICl11DNA
~-
...
~ I JII
,..,..~... . ... C
~..
la 118.
fil
.~
......
III.. III. -"....
l7W"
IAIMI
III
...A ..
871
el.. MI.
II
J.
11ft 'II Pil"""""
&
fil
.. ,
11M.r
II!'ItH"
..
m
la CII8....
Alt
.. '- ,.
... ... ., linI~I.M
~
l
tir ...
D4..~~...,
.
L
thII 1M
..."n ull"""
8
J1
1a"8 ..ut.-.,
ful III ...,.
IIR~.'
el -~
... ....-. ...
.... R."......................
- 8M.. . ".. . .'..-1M
,NIuJ~IIJI
pI
Af'it
11111
IIID......
.. redlII8M ..
lit
~
.'II1 III '
~
1/.
pl\8..fW16.
o '."'.--"""""""L
.. -- -,r
178 fil tII8
.. . ... ... .. .. D
r
HIn
... - L rI f8r
ilie...
,...
L
f.
... .......
TIIII- ....
III III ........
- ...
I' IlIlAllh n
r
II
171...
.. ...la
,-
. "'W.
.. de8cribe- ..(PCIl)
BI8 ~ lIIIdIod... .
MilatWO
~
-. wtda
poIJIII8U8
tb8 ]"'"
..
JIDOt)
_1OOntId.88
~ 0lIl or badaprim8I
l8JIIIIJ dI8 RIa B or lUs.
8DeI8 (8'I..I...Ip"'{'IAe
ftc8daa
(ASPA)].
W. .-L..-I
8D
pIiD8.....
--- tWOPCR880~
rn.
miM... RIa
B... .. . JIDCIC7P8I.. DNA
.,. wbI8II...
~
wId8e~
b8d
...
bIDodcbIon dill
pb8aoIyped tar RIaD. RIal!Ie, lIId
RIaCA:.
MA1IJIAL8. AND MITHOD8
,
bl lIIDod
CIII8..
- [...~
.58""""
~"""a.._"Dp
"D~(IW
(1I'8I8b8
.. lIIi
""')
B,
a.8)
C. ..
. .. . ~
lo
...
D.........
a.a,.
fI.-.
- I. I ("".
r,Y, C
a; . i r,M.
---' -
C. ..
.
I
- --- ~- L -- .. .
'j ,lir..
... 'j III ,W 01
.., ..
CIuIa . aL 18
To
.
-
..
la
lHpIc:IIa
.
ASP
- -_o
.~(~
,... ...c...IlM
) ~... ~
I . ~ o.. lI1IIItI
..,. .. rA.
.r..t
J"'>
/tir ...,
.. ~
~JI
JDo~~A'II
"""""'~4.
..,
~
.H.Jllf.dSl_.
~
,..-.
,...
c..r. "", "U
,Ä lIir"..,.
. ~- 2J.19!N;
.,.,.,
0tåIr
.J. 11M
,.",." ,
. C& ,,".fdIIIc
lID. 1t8D,c..
...~."..
lIå. '",. W O...,
~
IOIJI er A ]f ...
n. NJ6j I A.
lIir ~.Au ~ II aMII
.".,. il ,." ., ,...
INIl.t
..,.,
...,
"'
'*'" ,., ...il7IIIr
... ...
,."
..w
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" U.s.C
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1114"'.
,
..",.,.
O 1- ., .",
~
~ Il , hD-
fJDtJI.IR
IlfSlllSOJ.oIJDtIIJ.
'*'
Val
......I ,
11.
- ..
-
..
fAlla ET A&.
,.I nI""
TIIIII'. L111J "~1.111" PI"1 1'-
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~
lo
fil
T..-
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CD..cDI
.
AlM
-
2t
II---31
co
ca,"""'"
c::D.-cIlIl
17
11
cOI,4a8
I.
aDMe 1'1
/I ,100..4II18II
cDIJaDI
--1141
.
31
c.-
10
..
11
cI/O8
I
CM:I
CDM:DI
CDIMJI
naIVP88-
pa8dCI8
-
+
--
-
+
-
-
-
+
.,.
--
+
--
+
+
....
-..
+
,
J
+
+
+
+
lir
... flmlly .........
...
--
--
..
IM ~
.
E allele 01 me RIa CcEe
..
( _mi
~ becIUII of die
...
01 me 3'-- ..Jeotide 01pr;... R661c. wllicb il spcciIc
for nuc:leotide676 0I1b8 Rh E aIJeJe.SeeB ampli.. . ~
--
lo«
...
-
...
b8Id w. llill viii... ch8IPti die MA concealnliOll8
rauJ8edla lb8 bl al ilie e-1p8CiJc
II 1.0 nuaoUL
MtCI1. Tbua.lb8 lHp8CiftcASPA i. opCimallt 49"C........
ini boollper
1I8int U nnoUL MA.
s. A ampW1e8 101-.. pIir rep. lpOCiftcfot die RIa
....
-
lo«
...
---
......
- ..
8D547). To p8b8
betweeD46-c lIId 6rC. bIIIdI wereal..,. obcaiMd.
How.
ev".II lower
~.
lDDI'8
UtlKlftc
WW8
....
A hi'" concentr88iaa
of MA resuhedin ICIOIIprb8nd8.
The opdmII 00DdI
119Mrar tb8 B-1p8Ci8cASPA WtnI ID
...aInI
l8mp8I'I&IU8 uf 6re 8Dd1.3 rnmoULMA.
1be
CQfIIhI...ritwo( pm.
B lDdC onI1pw aoad~
lit lUllle8Ji. t8IIIpU8IIIr8 al WC 8Dd49'C. At hi..
Ibee-eped8c .. lO8&"
---
..-
+
... -'II.""
... ... i8'I"""'~~'"
--
...
181
,.
+
.
,
.
.
......
AlM
II18II
11---
c::D.-cIC-",
-.
~I"'.
.......
+
+
+
+
+
11
""'1
" 111M
.. ---.
!!!II
(- ..
ASPA.B
wI6 l8t C: prinIIr R661,
prin8:
"CCAACmlI'CAACI'C1'O3',
paIida8661ID 676)... prtRIOI
(-an--.
,'CATOCI'OA1\.;II<A..wr3',
101
ID71'7). Al
DNA.,
(Ap-
-.
lo)
pi'" .-,-IIIDdII392.
No Al.. CA),
R661..
.. ..1 ...
c:8ItItd88
(AfpI6Id --.J11
'I1Ia PC8
...of-.50IL naA...wi
. 8DI8Ivoa.
... ,n... 0.2~
IMIe.
. crt.prtmm
puri8c8åa8
.'"
C!IJ, CA).
0.7 ". fil
DNA
A
..
75III of
of
cINI'P
(,,*,. '. UJII88I8,
S-
:u ~
M,a. rar... B-If8CiIcASPA
d88),1 U of 1'aq DNA ~
WI),
wI6
18dia
....-
(""
1.5l8IIIIIM.
MA ftIr"",,-,1I:
wIdaa DNA ...
c
9S"C.
... 1.3
(~(
-~.-I
4IQ,
ftIr 1.3
al
ASPA.A
CXIIIII'aI
...,.,. l8CII1dad.
"..,...
~
of
.. .
eJder (IWId8 Bb8ar
CT). widI to
,jII~.'. I
al
-
-
.. 62"'C
rarpdnIIr
A... .
.wc rarprtm8
B ... C.... .........
r. 1.3
al 1:l"C. 1118pnIdIIc:ta-poIyllClJII8IWI
..
, ~
CI8
. I~
.......
:!!
.......
~
lO.
)
REIULTS
R66lc1R768
(lit A).
A), R4871JU68
R41711U68(lit
<lit 8),
8). lad
....
B
R661tlRJOl(lit C)
de8ipecl
8iped CD
IDampIify
lIIIpUIJ 1pICi&
8p8Ciftc
...
810lIl
011111
ab CcB88DdRIa
Same
.D
D
SomeeaperimaIII
nperimeDcI
PlI t. t 18u
r J1~
I
el.. MPA.. ... . ... ... .
were bmed lo 8'l"ive
. !be
lIJeopdm8I coadIdoaI
coediå08lo10...
III
...
.....
the pria8
PCR81WI1I .-tu
ecI
widI
__H...
lemIII&"'" wiIb
ra.
.
.. ... .. ... CII8 ... ... ..
~
of wc. 49"C.5-'-e, ... 6TC. MA II'a
III D
... . --.. ... . . ... . .. el ..
III
CI88
IN
r'
r
~
el
.. ...
MM. III
doaaMR vIried
Ihe opdm8I
_H.
~
ipdmII --un,lIeoaper.duN
A mmoIIL.
..
A_'. mmoIIL:
... A
III
(1.0 mlDllUL,1.5 8IIOIIL, 2.0
ud . 2-'
IXi le...
el..
.. III
MM. III
multi naI 1IIowa).The COIIIbiudo8 01 ~
A lad
..
c
I~J
le...
..
,
...
8 worbd weil bl . 1... l'8nJI
al __II.. l8aIpInIIU'eI;
el t.t .. .. - . . II.
fri.,...
...
~... 62"C.
MA
..
..
"-
..
...
.
elMI. . ,.
u
...- ..
"-
......
.
..
-
.......
. ......
~~~
~
.
8
6
M
-
zoobp
-
100
bp
141
~,-
101lip -
.. lip-
11."""C
1111/.
I'.'
... A 118M
II:""
,'- 41:
--
.., ..14
1Ipt. ..,II8Iv.L
III I. III
_I:
DNA""""".
J 8M t: DNA"""""
. ... . DNA
DM.
III.
Lo.-'
MI
..
8k lao.
8twll
.
PCIIIh
-""""""'"
aB
- .... 8
11JI C8G18
"'.
fil.. ...
bp I'Clion in uon 4 common lo the Rh D JCIICund tbc
Rh CcEe acne.The IOI-bp
and the 94-bp bud weN
,,-oampIifiedhl Olle PeR. In Rh E-ncpdYCDNA ~
eN). oa1ythe controI fraame. of CMbp la found. wbere88
in Rh E-posilhe DNA gmples (ee or EE). the ,,'OIIU'OI
hlment al weil u the Rh E-5pCICitk l08-bp fruammaareampli-
lied eFiI lA).
Set C IIIIIpliftn . 141-bpn:llon or lhe Rh CcEeJC11C
'lpeCiftcto the Rhe allele. Prinw R6611hM. 3' -endDUeJeollde (nucleoride676) 1- enabIeslhe primer 80biad 10the
Rh D sene 81 wen WI to the JUl e allele of lhe Rh CcEe
ICne. buanocto lhe Rh E 1111e1c.
Primer RIO. ... . J'-end
nudcotidc(at pcIIilion787)thai iA "f*itk for theRhCcEe
acnebut not the RJtD JCII8.Whea prim« 5etI B aDelC ure
u!led in the ASPA UI8Y. only the 94-bp COCIuol
frqmeaI il
aunplUicdiii Rh e-aepdve DNA sampla, wlUle in Rh epmliw DNA l8rf1IIn two b8adI ilie produced:the 94-bp
control bind ;Iftd the 141-bp Rh ~-~iRc fruamentlFiI
18).
All 01
of I"~
I" S8InpIe8
S8InpIe8
were~Iy
seroloJic:ally
pbe8otyped
for
All
were
pbenotyped
far
Rh Ch:.
Ck:.BI..
EIe.aad
a8dDD eT'"
eT'" ,).
1).UsiRl
Usm,tha
theKb
RJtSo
S.aod
lIDdKb
RhooRh
speciftc: ASPA. we typed aU 01... doaon for tbeir Kb B or
Rh e IJenotype. The mul.. of the E-!.Ipeciftc:and o-!.Ipeciftc:
ASPA and lite c:oncJIWo8i with repnI ta ... acaotYJ18are
IislCd la TlbJc I. Thetw w. compIete IJIRCII18nt with the
reMI'" of the lerotop: Rh EIe pbcaoIypin.. AJI of the gm.
pleti I"" were scroIo8kaIJy typed . EE or Be produaId two
III . . III.
... C... . ...
......
n I l..l III """'111
ilie ...,.-
C""
II
... A
A
III A
88:
... ..
~bcd 10dctcrmincIlie Rh D JCftCICYpe
at thepnomic: DNA
levell"u
In dw praenI 51Udy.wc lIICd two ASPA reactioM 10
detcrmi8ethe Kb EIe pIIOtype on JCftOCI1ic
DNA. To "de
ou, f8be.neptlwrad~
c:8U8Cd
by in..f'Bdc8IquaIity~
the rapt1b or r.Uwe 01 the l'CaI:oo.. hsclf. we inc:ludcd ID
inta'D81
CODUoI.pri.,.- setB. th8tahnys lives ... amptift-
clI'ion productilmll*'iw: or the pflenocypt.We perfCJnllCd
the raaio8I 011 DNA sampletfmm I,. votumeerblood
cJoaoa.vi'" diverse ~Iy
lletennined Rh CcDEe
pbenocypes.
The reeaJlIof our Rb E lUIcIKb e lenotypin.
were iII run c:oacordance
with the multi of the scrolop:
phenoIypiltJ. By plMJlypin, DNA .ampta from donan wich
ove allclet (CDE and CdE). it was !lhown thai only lhe ca
"vmbiuhon.whic:his by f. IhcIl1Ol8
commoa.
but abo ilie
CE ~"nadon c:oukl be IlInpUfied.
Rece8dy. Hy""
et II'" UNd M. I RfLP di_on
pMteml ~ the )' noacodbtl repons oIlhc lenea lo dctmnine
Rh EIe pIlot y... For e Ihey showed a I ()()II. c:onc:ord8a::e
b8tweaI dill laUIb oI-mop:
~
lIId acnotypi.,
b8ud (8 RfLP
Id ror E the conc:ordanc;e W8 onIy
94.3".ne disaepuclatbeyCou'"bd... d» resultlof
seroloP: phenotypina and ~uJ.
JCD08ypinl appelftd
to be ~
wida the cB alJeJein D-neptive 5UbjectL
The. ca ilie- i. Cour
D-neladve
donors
whoN DNA
W88
ca8III
""'
. I:e.
III JIIDOtYped
No di8ctep8acieI were
bandawhell usJnl primer !IetI A lUIdB i. theASPA ~~.
wlleaIlie ca aj" occurrediII . hapIoIyP8
with theD
ane. Pmmme. rauIII. the InYftdpcon concluded
th8&
cc only producedtU Iower band In this "*ti08 (Fil 2).
This p8dem WIll round not oaIy la the combln8doaof E
me ca aUeleIs IOffcrcnt in the pmel8C;eor Ibseace of the D
SC- However. becaaIe chey were only UJalyzina I1OIII:OdInI
rep..
tIane rall'"
oaI)
becween the M. l
whcreal
.1101thesampla
... wen~Iy
Iyped
.
with C. bal ubo or E with C (!Iee the nn CDfJCDE. CdfJ
CdE aad CDEICDe pkaotypa). All ~
tUt wae sera100icaUy IYpedu & or ce plOchK:cd!Wo b8ndI whea primer
'leta B lIIId C wcre !lied in Ihe ASPA: lite RIt c:-~ilk
band ut 141 lip :andtlte conIn1I band. All ~mpla thaI wcre
~lop-aIly
Iyped III ER only product:d the c:onbOl band
rFiI 2).
DtSCUSSIOH
Until recenlly. il was onJypossible 10det~inc me Rh
C/c.RhPJe.1II8d
RhO phenotypes
~Iy:
buabecau!ie
.be ~!IC'"
RNA sequenca or the Rit Cc& and Rh O
lenes hevebeaJmeknow"," !lCYCnIJmcthodI hne beende.
.dJow
Ii..
RfLP al ilie RIt EIepbeaotyP8.
Moreover.
no DNA fmm
cidler D-poIiciveor D-nepri\'e subjects with the CE alleIe
WIIS Iyped. In the ASPA medlad. we Inled DNA o( !lix
donan containina die cB hapIotype ud lIe"er mc:ountered
a dllmpIUICY. Funllennore. we lI1to lälled !'IOtneDNA .....
piet en. 11ft phenoty... 1'hc!e remlb ~
ulM) i. full
coaaxdlnce. For RfLP plitan analyJia. 18IltJc quaality of
DNAil needed.
wbaaafor miaPCR-baedmcthod.II small
~
01
DNA is JUlfic:iaL Thil 5II11IIamountpuriJled from leM lhu O.~ mL of whole bIood or from
lIJIIftiotic c:etll-makcs anotYptn, umplea from fetuJClalld
fmm pItieof8 with S8veretnanII'usioa problcmareaible.
A ldIoutfalbe mulll 01 <MIl'RIt EIe JCOOCYpin, w~ i IJ
-
I' AM
IT Al.
NU 00IICGI'CI8IC8
WlIIIIII8 l181li8 UJ"U8I
- RIa
IlA!
1pu1lo
d8e
BI.
tbI
El. pbcaotypia..
caaconIIac:e widalb8 rauJII of
one rmJItbe awue of die f8ct dill cU8cap8IIdeI
occur
cIia8p8Dc"
oc:c:ur
jarW'8
c-. lUCb
. ~
ja wbida
lddId88
tb88il il~.....
cr---i..v.
wbidI
of .iIea&liliiii . the RIaJocua.
.IDla ~ C8I8
C88 (...
<...die
tb8
RJ8..8Dd!bl
D- pI8Iaa&ype8),_lUt. lU1
Cd!8,..1I
CcE8
... ilpnI8III
......
withouc .,
rarnnpmenII
lDIIIIdoaI
wtdda die
rarnnpmenal1lioror
06
fI'IIIUIOIII widda tb8
codiq
bal DOCJc or Ple
cacabebldeI8d8d
deIecr8d
011die erydIroc:ytea.l.
o. f8II1I ,*JM
froIn tbI8
poup of do8Jn COlIlInDtb8I'"~
88IOCi8ll--Ibec:yUJIiaeor auni. poIYJ8DIIIbiIlD . po8idoa 676 of... RIaCcBe... and
the Kb B or Kb e pbeootype.I ~
weIy.The8 ladInp
Sb'DD8IJ
th8t proIiDelIIId aJaiae areiDvoIvedin the
5peciJ1c
epi8Dp8tb88.. recopDed by and-Kb B ud aadRh e 8IIIibodIea,rapecåvely.
In c:oaduaåoa,
we praeat . limpIe lIId reIi8III8 PCR-b8aI
metbod to dItermi- the RIaPJepolymorpldllD OGpaomic
DN A. 11åI C8I bl uefaI to cIe88nDi8Ibe Kb BIe pI8OIypI
iD retulel iii ID euly pbue of pnp8DCJ and iD recady
prachadby""""""""
BIDad.1074, 1992
lr8Ifa88d p8åeaII wiIb
8IDOUIIII of c:imII8d88
cIoaor
9. Ma8a I, c:I*tI-z
CJ-P,lA V. Kl. C: ~
I..
b8IiI al
11- bIDad
.,-. ,...
cell..
-
0IaIdc8
al ~
0nIIIp Serolog
lIId Dr c.P. ~
tar
REFERENCU
I. LewII Mo.u.. DI, Bild Owo.
B, C.uoa J.p,
ea.u.. ... CIook88 MC. D8brw. D8Di8IIOM.
CP,
00. CM,
PI). -
I, JtQI8II8d
... l..uI8IIIb
A.....
WL. ~
J,
BPI.,ManI p,
D, NIow...u.- If.
Nof--- J .. Obbo Y.'~
RB.
PH.1hohI
,.
P.
WQ, WdI So~
p, 11ppII8p, WIIbr RH,WoadWd
(], YOll8l S: BIDad ~
1990
..~
1990. Voa SI8I5I:IS2,
1. L8wII MoAIII888DI, Bild OWo. lInIIII8a B, en.. J-P,
l:onIIw8 ... DMr W, DI88II aL. ~
CP,I.. lO, ,.....
.. J. ltarDIIIdL. LuII8dr.oA. ~
J. Mcal P, Mo8IdIU,
-- If. NI.. .,.. RoObbo Y.
J.
PlI,......
I". P, SIIaIe801, SeIdlSoS
P, 11"" P, W'"
JtH.
-
IV aocMIIId
loIou
H:I.58,
a. y-.
rar lid cell
1991
So ZeIIIIIId T: lS8T W CIItia8 ,."
..,..
II1II1181:
Lo8A8piIII'IIpIn
-
- ~ -- --
S:6Z, 1993
IQ....
ACKHOWLEDGMENT
W. dIa8It... rall alibi t' l rre. h8Ip ,...
d8 bIaad
CQI7 I .. !be~
I-r,--- I;
-
......
-RH.
....
VCIII SI8I
Sodl -. CAN,
BIeIIr8r W
1M,
Golc'o""""".. ... YI8 dir ScIIaaICB, 0djpIn RI1IM, VG8
lon8 ABOItr. s.,- ,.
fil c:DN
A c:-. cIIrIwd e~
o_.
,~
mRNAa c:odI. rar ..
pot,~
Voa ,..
...
67:-
II. ~
PR.La V-KIm C. CoII8Y. WlrWIckaM, PIlla
PRe.~
.. PIK NU, c
UJ:
............
al
RIIDtyp8 .., DNA~
N'" J Med329:d07.
1993
- -.
A "'u_-- ~-~--
t2. ÅI'CI MA. 11IaaIpIaa
ss. w
S. ea,.. KB.PwIb8
BA, LII1IIt8 DM: MoIIcIIIIr cIaaI88
01I0Il) cDNA dldftld lra8
... ~
la ~
tJutnat lUll)
~
.
11- bill."'"
BIaod 82:65 t , t99:1
t3. S""
S. 8IIIår PMM. ". ..
ABOXr. .......
01... DO typL N BII8I1 Mad 330:195, t9M
14. Hy"" CA. WoItIarLe, U- Y-W. SaIII A: A Sa8dIam
lII8IJI8 olD bbId palp
..~~D8
tcda8
/'rapII8I
poIy..,... pI8IInI... .. l8'OtJpII.
BJood
13:jf6, t9M
15.CIuI8TA, SkIar
.-- SI.: A
far DNA
lra8 plit,
bIDad.A88IBIDcIII8174:415.
tt6. CWdt-ZaI8"..,...V,c...IP,CaII8Y:o..
' '...
II1II
01die .. laaIala III ~
widI
,
D- - ... DC- ~
Jr J
17:t
19M(...,o
,
4"-
~
\(
~
The presenceof anRHD pseudogene
contJtining a 37 basepair duplicationand
a nonsensemutation in Africanswith theRh D-negativebloodgroupphenotype
B8IInd8 K. SIngI8an,CII'OI8A. ~.
HIll D.Awrt, Pe8r ca.M8rtIn,EIDb8Ih Snwt. Abig8IID8Ic8,EdwInG. N8rt8r.ol8g8,
Und8 M. II8wIt1am8,lIId GeaI D8nI8II
.
Ek . .'"
MIc8nL RffDItI
oanIIIIn8 Jr
.,......
4,
...,
encod8d bf 2 tIMNIlala..
lI8ft88, b88 pIIr (lip. ln8IIt In . .. codan II
211.
RHf2,
~
2 r8d C8II 1ntraduc8
AntIg8n8fil the RIt bIoacI
..
RHD
...
rMmbnI18
notyp8
.. CGI""""
1118
D-n8g8tM
,..
lo
lImo8t
The ~
lCI'OI8
..
.
baundIry
-
--'lir
oIlnnn
l~lft
3..
4..ltIQI conIIIn8
codan In
Im"""
hm homoQIMtIr far. ~
.. The,
oIlIID8Iln bI8cII
pt8II8d8I8IIonvi RHD.".
vi ..
SouIIIMIc8n8 .. lppI'OIdIn8tIIrU714..
PCR
far ","".
D .......
blick AMcInI,
hId
type hin DNAobI8Inedhin 8In.1IoaytiI8 Ofa Ek ".
1ft hId
RIOa-D hrbrId
or
.. d.lIlI""
fil the ~
108vi RHD."
U88dIn
ord8r lo .. aI r1IIIn the rt8II fil henIot;ItID
dI.-
((8
lied
vi the
.. RHD
... .nn"'" wIIh vs+
~.....
typ8,.. onIr1ft compl"'"......
Rh ia . hlpIy compta red een blood poup l)'IIan wida 52
ad...
ad ~
pbeDotypI8.l.2TbI Rh 8DIipaa ...1IIIXIded
by 2 c_ly
liIIDd bamoICJIOU8
e8da c:oaåI8iq of 10
ac8.
RHCBeacocIeI
... CclIId Et 8DIipaI, lIId l1HD
aDti... ThIIre an allo mall)' UDCOIIIDIOII
lb8ioa
p8It of RHCE lDd plit of RHD, wbidt may
D ad CcEe
lIId l or lDOI8Jow-
-
D-n8gIIIM
III"'"
.. RfID8i,
...
8IIIr rHUII 01 ~&"18d
mRNA -.
!Id88Ing
PC~ ....
-.
c* tar
...
dla1IrI8D pe.1Otype""
nae
for
DM
,.
At'Itc-.
. ..........
""
001""'"
propoItIoII01
MIc8n .......
nr.ean
,.
lI8W
o
we
lh8I
4 01"""..
-
--
In
24,. fil
We..,. Id8nII- RHO.""". le
CRHOIIrtIn RIt f).negIItNMtcIn AnNrtcII... 1~ fil
Introductlon
D-n8gIIIM 8outh AfIto8n8 01mI8d r-.
No RHD ini""
cauId b8
III
..,.,..
.
IM 37 bp ln88t III
(1Iood. 2IGI:.:1 ~11)
lir T1I8
AII8tc8II
011- II aIa.
pheDotypeia peaple of AfHC18 orIPL.D la 130 D-nepdw
36(28%)h8d- lppInIItlyiIact RHD,2 h8d.. lcu8
1
b8d RHD C'JUIII
3.11Delpitl
of 1tHDis
tbc1IIU8l
moIecul8rb8cqround ror die D-neptiw pheaotype.
tbe8I
10 of 1tHD,
l 0(
allo
vma.. bIrMrYer, il is .-.DY lCCCI*ddI88/å
1beD
isofcliaicalimpor1IDce
--
i. .. C8p8bIe
ofc8a118'WII8
lI8Wbam(HON).~
ati-D "i~
bImolydc di8eue of the ...
II1II
ia hydrupI&talU II1IIfeI8Ide8da.
ra
lbaIe .-ly
iaUIIaDwbo.. bomaliw,j8UDdice
may
1- tobmic8Inat21be
JllWYIIeDce
ofRbDHDN" be8 p8dy
reducIdlila ilie l ~
by ilie iDInJcIDctiaa
of IIId-D ib.b~
liD pnIIJIayIaiI. whicb prev.a D-aepdw modIIn &om .....
i.mIm8Izedby fet8ID-poIåtiw red coOl u . reIUIt of ~
bemonIIap IlpålritioL
SameD-aepdw WOIDIII,boweYer,åiJl
.
IIIti-D ror
VIriety of reI8OIII iDcludiat lIDde8ected
t1.4 feeo..m8naIlIemonII8p 8Ddfail... to IIdmiaiIlf8r
_-D, 8DdIIIY lUb8cqueII& D-poIitiw f'etu8 i. riIk &om HON. It
il DOW COIIIIDIIII prKtice ror 8Idi-D immunoaJobuUa
to be 8dmiaitprocb:e
.
, 1(
\~~,'
. tf0
. !
tered uy
.. weR.. poIIn8t81ly
iD - lIIempt to reduce ilie
rilt of D I_MtiDiacovwy of the moIecuJao
bIIia of ilie D poIymorpIJic:n8tId die pollilliJily of JII8dictiDItiIf8l D p/JIaoCype &om,.,
DNA _ved &om lIIIIIiocyteI obIaiDed by lnIIIioI.'eaIeIi. D
p~
of ilie recu. 01 . D-neptive WOlD8ll
with IIIIIi-D
provicIeIvllalble inform8Iio8fiJr p:edic:tiu,the ri* of HON lIId
-.,-
ror lUb8equeat
o( the pntpllDC)'. Tbe fint teIII to be
-
FramIle lIrtI8aIII'IIIIUI far 'TI'InIiIIIan ~
1IriI8aI,EngIInct tII8
InIIrnIIIoIl8I 8IDodGnIup R8IIII88 L8bcnIary, 8riIIDI.EngInI; tII8 NIIII Ra8d,1Irt8I.10 5ND,1ngIInd:Hllll:1IIGI.dIIII1I8.III..nII8.uII.
8IDod 1'r8nIMIan lI8nIa8, PIn8IDwn,SoIMI Mta8; tII8 lIIoadTr8nIIuIIon
The puIIIaIIIan CIII8I
cIIhIwed In plit bJ Il8Ol cfI8'gI
S8Ma8,
Z!nIIIIM8; tII8
lIIoad,.,
lI8nIa8, Aan,
~
TIwIIaIe.
Ilet,
'*'lIr
0hIn8; 8ndtII8LauIIIIn8
8l1liUrMr8IIy
C8nIIr.
~
lA
rrwIIId
1iIMI8/11"
In_dille» .11 U.a.c.--.1734-
01
.. ...
... ...IDlndIc8II
..
SubmlllldJun83,
1_""""
"",ob.. Z.1_.
SuppcII18d
lir . .,.nI110m
DIIMIdNJ,CI8IIIIHur-MInI.~...
12
....."
C 2OCIO111
n. Am8lc8l1IocIIItr
al HImIIaIDsIr
8I.OCO. 1 JN«JM(
.
2IlOO VOWMIII,
lIU88R
1
IILCQD, 1 JNIAJNf'f
2000
.va..uue.. NUM8R1
RHO
... ~
INAHD-HEQATMI
APfUCANI
13
devi- åaYoIved
a polymerue chIiD reacdoa(PCll). The teIt8
tb8pnI8IIII8
or
oi.
cIItIIrmUIed
q\IIIICI8 ja _oa 10 or GOD 7 or nICOpized
01/UI1Hpetcitc
.
Ibe.. cIifI'tnac8I
betwe8iDIIuDI 4 of RHD ad RHCB,I).I'The8etec:baiqu8...
t"oaIMt
to be wnli8bIe.l' The 8bIeac8of cert8iaRHD rep. iD
encodia8pIIti8I D
IIIIIJ predIct a D-aepIiw
pbIDotype,ad dia ..of lOIII8RHO rep. la ....
eacocliatDO D -pli
may pndIct a D-poIitivejJbeDotypL
la
onIIr 10avoidtbnI compIicaticllll,mcdIocI8
w'" man
l
rep. of RHDil cIet8cted
iDa ..
PCIl w.. iJdrocIuaIcL
6,11,17
hybrid
AnIaioc:aaIiI
ila hiply iDV"ve procedur8 8IId DDt witbout
It 11181 allo lIIIhIaceIbe riK of lDII8InII
imm-iw8tWt
Metboda lave bele de8aibed rar precIic:tiIIJ D
phcaotypI tum fet.8J DNA ia lDIIIIm8I plumL
It ... bee8
propoIId tbIt tbnI
tecIIDicp8 couId be lIIId rar taIia8 aU
D-aeptive pre
WomeD to
wbetll8' tbcy requiI8
riIk ID lb8 fetul.
admiaiItrldoaof
_-D
~
wouIdl'lllllal'N8I8Ipoflbl8val...
nIOUI'C8
11,1'Te1dn8
by avoidiq uti.D
illll1l1llllJliolia 8dmiaiMrIåoD
to WOIII8
wi8b . D-nep8ivefeta8.
rar predIctina D pbeaotype hm DNA or mANA
areb88ed
oa tbe dopIa tb8t,willaoaly vrtty rue u.cepdca..Ibe
D-aeptM pbIIIotyp8 reRIt8 hm tbe lIIIIace of aU or lIIOIt of Ibe
RHD
MoIt
bowwver. have bola p*lbmled lID people of
Europ8ID orip.. ad the molecul8r t.ia of D poIymorpIIiIm hM
not be8 8decr-'Y
iDveItip8ld
iD otb8 r--.
pIdicuIIrty
iD
people of AIiic8D urip
W. have tn8ed rar the preMIICII of tbe
RHD ...
ja AtticIa8 ..
AJiic:Ia Amå:8aa ad fouDd ... tbe
~urity
of D-nepdve AIti~
8Dd about a quuW of D-nepdve
Aftica Anaaic8u have - ill8Ctive RHD ...
willa . 37 b8Ie pair
All ~
-. ---
(bp) u-t
RJlD.CB-O
... or. ~
mutItIoD;tbeJ'IID8indIr
haveoi.. tbe
deletiaaof dieRHD....
M8t8rI."8nd rnethoda
--
~
aaIIIawII
au. . ~
blå ScIu*AIdc8 wI*...
~-...--
a-
ltHD 37 bp
~
CPD~
\AJI~""
Im .,.u.e; SI.-AIti:II. PooJe.
l!D8J8d).
II1II
GIlla--
.,
--
(MI8I.MACI
1ba
...
2 CJ'I'Iaa
till
BIood
......
((~~:
..
rr-tpe"'"
la
MIJI8,I"'"
cbIaridI.CUM+~
a-. EIIII8d)
'"
-r-'
ilad 1..-
wilillIIIIia)34
wIIoI8 bIoDdiD
-- ... cuItInII
il .....- 01
,
~
......
-
>.r« _iDIIII -(rH)81)""""
(JIoIII.
o.aepdw biocid
-- obIUI8d~ bI8drAJHc8bIaad..... (N.v.IIJCb8,
rIJIIIr
'... FråII8t, 0.-,),
dl ~
coIo8y
hD 9011*
AtrIc8,
ZImbIIIwe.
... o-.; ... SoIåAAie8
al
oI!-~
lIc8ar
(UD
9ya8aa,
AIIiJIIda8,
Bn8Jand),
lIId
rH
~
mis8dr-. ndIImd tD "CoJoInd" 18SoIå AAica; 111111taIIIAttc88
'" MaIiklit aLD
A-'~
die al ~-.
1taIII
SIIn._1, ...3 (IL-])~ Sy8a),. cIa8arib8d
AIW II cI8J8,
ceIIa-- l8n88IdlIId
lir die ~
till
LouiIiIM. 8Iood
hD l)..pIIIiIM bilat SoIå AIto8aI
~
A (OPA)
.. RhproIIiIL 11118 ICCG8lpllIIIed 1IIin8
dldwdha -IIUIIJ..
All
biocid
coIIed8II iDID .
citn".t"jL~t
. .h..d.-(aD)~BIood III8IIIbrcytI8IIIIJ widt - 1lio8ii lIIIIIodIII BRJC ~ lIId BRIC 69 (UKW.
BrI8I. EaPad). BRIC ~ cII8IcI8.
- ...
~
~
-- dieby-pIoducI
albIaad ... -
.-
--
-
--
tD die approvedpnJIDIXII
01... lif8DIftI8aa
coI1ectIII
iJlftIIMIL
ww.
albIood,
.. ~
,.
--
widI
iDIxmaII---.
- r ........
,- .
-
CIIdIIDIIJDIIr"'"
..
....
.. BRIC
69
--Jular
lUaDpa/JIpIpfIdIL About 809601... c:IIII8
bodI Rh """'II1II
aPA.
WI iIoIaIId IIIRNA fnm 101ceIIa(1be PoIyA'nnct SY*D 1000!dl;
PrcnIp). JIIna.8rIM cDNA !lir rea .""'111 .11118
widI
ID
till GPA.
-...,..
.
... RHCcII8
lIId
--
01'" (4T..)."""'"
m."""""""
vfn8
~
tbr D. C.o,S. .. G,vs. 8Dd1Ul32........
till (PII88dI, MIIat lt.IJI8, Bap8d). D cDNA -1IIIpIHIId
wtdI die
vs+
aI8o
tbr V II1II
S8nJIoaicaI (III 457-476,
pedIn8d
by
...
lIIIIbDd
JIIO8t
tD ... pardcuIarJ'IIIIIIII: dInICt fbIJoIriIIIpr-..: 5'.aGAOOATOOTCATCAGrAA-3'
,,---,1ItI88tIcII.willllI8Iheor~
cIIIJI, .
All ... eeO
ar iIIdINct
laG(BPL.EJ--.
liDada8wllIa
C8II8 by .
kDowIIlD I'118III wItIa l'1li
IIIIIW (I!1Mit II, O-
...
lIoIIcuIIr
,...
o.-
DNA
DNA
ltc:IIIoa
~
kJt
..
..........
-
l!II8J8d). All I).aepdw red
witII 2 paI88 IIJoIaIi-Ddill
--
.. -
01111pIId8I D pbenatyp8LAll lCiHIuIiG8
HauI8a8,
TX)BioJoP:aIa,
3 till lHCIl ...
914-891,
tbr
--
5'.QAGATCAOCCCAOCCAC-3'
bp pI'CI8at
...
(III
~
la addiIIa8, . l/lD.tp8aitc rea wiå . pm- il ... 3 (RHDEX3', 1'IbI8 I) ... .
" -- 7 (5' -ACCCAOCAAOCTOAAO1TOTAOCC-J')
. 645 bp prodDatwiIIIlHD cDNA. Al . CCIIIInII. . RNa""'"
II1II
wItIa 8111i-D.
to
on g8ftOftIIcDNA
CAOCOOO-J')
rea _-W
.--
OIIIwiIiI.
- pn..1a-- 3(5'-'J'COOTOC1'OA1'-
...
~ la
. .. 7(5'~TCAT.
to81" . 715bp
wiIIIllIa aDNA.
........ 0CCATI'QCC01TCX-3')
Mt'II " ~ RNA!hin. D-nIpIM bIaak
SaadI
AMc8
abIIoI pIUC8Ihn
...!'08wIIoI8bIoDII
wiIIIl
+.. -- 10mL01""'" buIJIncIw'". ull,"
,
WI).reasm- ..
la '" iIGIIdIIICD71
iIolIIId !'08
("-P.
RHl1) ...
6 tIllJHa ... nID). 1ba..,.
c:eIIIby I