The HLA-DR Microtiter Plate Oligotyping Assay: A one year experience in related bone marrow donor searches

59

IV-65 Willem Verduyn ~ , Anholts I , Jos

llias

J.M.

I.N.

Ooxiadis I ,

Drabbels ~ , Albert

Jacgueline

Naipal ~ ,

Joe

D ' A m a r o ~ , R o n a l d F. S c h i p p e r I , M a r i u s J. G i p h a r t x , G u i d o G. P e r s i j n = , and G e z i e n a M.Th.

S c h r e u d e r x , for t h e Euro-

transplant Tissue Typing Laboratories 1 D e p a r t m e n t of

I~unohematology

2 Eurotransplant Foundation,

and

Blood Bank

and

U n i v e r s i t y H o s p i t a l Leiden,

The N e t h e r l a n d s

Proficiency testing Application

of

in

the

Eurotransplant

biotinylated

oligonucleotides for

sequence

region:

( r e ) t y p i n g of HIa%-DR

T i s s u e t y p l n g for c a d a v e r i c o r g a n d o n o r s

1991

to June

H L A - D R t y p l n g s of

is d o n e up to

were compared

1992 s e r o l o g i c a l l y

1052 o r g a n

E u r o t r a n s p l a n t by t h e with

d o n o r s as

typing

= 44)

the

The o r q a n

fragments

and

biotin

(PCR-biotin-sSo).

donors chain

labelled

T h e m e t h o d c a n be u s e d

a

e f f i c i e n t l y to p e r f o r m HLA-DR t y p i n g for of i n d i v i d u a l s .

(N

novel method using polymerase

reaction amplified oligonucleotides

r e p o r t e d to

results from

Eurotransplant Reference Laboratory. w e r e r e t y p e d by a

In the

defined

individual donor centres DNA

large number

S i n c e no e n z y m a t i c p r o c e d u r e

is needed,

a l m o s t 100% of the S S O - m o l e c u l e s

u s e d are l a b e l l e d .

addition,

i n d i v i d u a l S S O can be

the b a t c h s i z e

chosen according c o n t a i n e d ~7 control.

to t h e

for t h e needs.

oligonucleotides

Per b l o t i0 w e l l

The biotin-SSO including

results revealed a concordance the d o n o r c e n t r e a n d antigens HLA-DRI

discordant specificity.

of the

90% b e t w e e n

a

the

the d i s c o r d e ~ t

in the HLA-DRS,

w i t h DR6 as the These results

is s t i l l

cadaveric oonor typing

A comparison

r a t e of o v e r

T h e m a j o r i t y of

results involved specificities DR8 c r o s s r e a c t i n g g r o u p ,

HLA-DR serology

set

a positive

t h e r e f e r e n c e l a b o r a t o r y for

- DR10.

In

defined HI~-DRB DNA controls

and one b l a n k c o n t r o l w e r e a p p l i e d .

A NOVEL, QUICK A N D AUTOMATABLI~ HLA-DRB TYPING TECHNIQUE, USING MICROTITER PLATE REVERSE HYBRIDIZATION C. P e p o n n e t ( 1 ) , V. S c h a e f f e r ( 2 ) , V. L e p a g e ( 2 ) , J. A l s a y e d ( 1 ) , N. M o n p l a l s i r / C a s s i u s d e lAnval(1), D. C h a r r o n ( 2 )

specific

d a t e u s i n g the c l a s s i c a l s e r o l o g i c a l a p p r o a c h e s . period July

IV-66

DR6, and

predominant

indicate

reliable technique

A reverse hybridization method using rnicrotiter plates has b e e n d e v e l o p p e d f o r HLA-DRB t y p i n g . 43 DRB1, all DRB3 a n d DRB4, a n d 3 DRB5 specificities c a n be t y p e d at once. B i o t i n y l a t e d PCR p r o d u c t s f r o m t h e s e c o n d e x o n o f t h e HLADRB g e n e s a r e h y b r i d i z e d to o l i g o n u c l e o t i d e p r o b e s c o v a l e n t l y l i n k e d to r m c r o t i t e r wells. H y b r i d i z a t i o n is p e r f o r m e d f o r a n h o u r at 41"C followed b y w a s h e s a t 47"C f o r 2 0 m i n u t e s . T h e d e t e c t i o n uses streptavidme alkaline phosphatase conjugate with a fluorescent s u b s t r a t e . O n e h o u r is n e e d e d f o r this s t e p i n c l u d i n g t h e r e a d i n g s . A l t o g e t h e r 29 p r o b e s a r e u s e d as well as a p o s i t i v e a m p l i f i c a t i o n c o n t r o l p r o b e . T h e c o m b i n a t i o n o f t h e s e p r o b e s gives t h e DRB1,DRB3,DRB4 a n d DRB5 t y p i n g . 8 p r o b e s a r e allele specific, the 21 o t h e r s h a v e l a r g e r specificities. DR1, DR2 a n d DR9 a r e t y p e d b y a c o m b i n a t i o n o f 5 p r o b e s , DR3 a n d DR8 o f 7 p r o b e s , DR7 a n d DRIO o f 3 p r o b e s , a n d DR4 o f 8 p r o b e s . For DR5 a n d DR6 the n u m b e r of p r o b e s is m u c h l a r g e r w i t h 11 a n d 17 p r o b e s r e s p e c t i v e l y . The d a t a c o n c e r n i n g t h e 2 9 p r o b e s as well as n e g a t i v e a n d positive c o n t r o l s a r e d i r e c t l y a n a l y z e d b y a c o m p u t e r s o f t w a r e HLA-DRB t y p i n e is o b t a i n e d t r n m e d i a t e l y . In c a s e of a n I n d e t e r m i n a t i o n . t h e d i f f e r e n t possibilities a r e g i v e n w i t h t h e i r f r e q u e n c i e s a c c o r d i n g to e t h n i c g r o u p s . I n t e r p r e t a t i o n w i t h o n e o r two d i f f e r e n c e s in t h e p r o b e c o m b i n a t i o n is also possible. In all cases the final c h o i c e is left to the o p e r a t o r . 4 0 h o m o z y g o t e ceils o f t h e t e n t h HLA I n t e r n a t i o n a l W o r k s h o p , as well as 1 1 0 h e t e r o z y g o t e cells w e r e t y p e d w i t h this m e t h o d . T h e s e HLA-DRB t y p i n g s w e r e c o m p a r e d to r e s u l t s o b t a i n e d w i t h a d o t blot o l i g o t y o i n g m e t h o d . No t y p i n g d i s c r e p a n c i e s h a v e b e e n d e t e c t e d for the 1 5 0 s a m p l e s . This m e t h o d is f a s t , 4 h o u r s f r o m s a m p l e s to results, sh-nple and end-ely automatable.

that for

if p e r f o r m e d p r o p e r l y .

(1) GENSET 1 p a s s a g e E t i e n n e D e l a u n a y 7 5 0 1 1 PARIS. (2l L a b o r a t o i r e d ' l m m u n o l o g i e et d ' H i s t o c o m p a t i b i l i t e , Hopital St L o u i s a a v e n u e C l a u d e Vellefaux 7 5 0 1 0 P a r i s .

IV-67 J.-M.TIERCY', E.ROOSNEK ", D.SPEISER *, P.AIJJlqERT +, B.MACH*, MJEANNET"

P.CROS +,

*Transplantation Immunology Unit, Division of Immunology and Allergology, HCUG, Geneva. +bioM~rieux, Lyon, °Department of Genetics and Microbiology, University of Geneva Medical School THE HLA.DR MICRO'lITER PLATE OLIGOTYPING ASSAY: A ONE YEAR EX]~ERIENCE IN UNRELATED BONE MARROW DONOR SEARCHES

A major limitation to the generalized use of unrelated donors for aUogenic booe marrow transplantation resides in the accuracy of HLA matching, in particular for the allelic polymorphism that remains undetected by standard serology. The extremely high level of HLA class II diversity can now be resolved by the powerful oligonuclootide typing technology combined with PCR amplification. We have developed a rapid non-radioactive and highly discriminative HI.A-DR oligotyping method that is hased on the use of oligonucl¢otide..coated microuter plates for hybridization of amplified DNA, followed by a semi-antomatod washing and colorimetric read-out. The assay allows the diseriminahon of 32 DRB specifieities, including subtypes of DR1, 3, 11, 13, 14, 15 and 52. We present here one important application of the microtiter plate DR oligotyping assay for unrelated bone marrow donor selection in the case of 30 consecutive leukemic patients and their 103 potential class I and I1 serologically identical unrelated donors selected from international registries. The microtiter plate assay detected additional HLA-DR incompatibilities in 51.5% (53/103) of the serologicallymatched donor/recipient combinations. Twenty-eight percent of these pairs were due to serological mistyping of the donors (DRll-DRI6). whereas 72% of the incompatibilities were due to low resolution of the serological technique and essentially consisted of DRI, 11, 13, and 15 subtypes known to be relevant for T cell recugoition. The microtiter plate-matched eombioations were further analysed by SSO-oligotyping for the DR4, D R l l 0 1 / l l 0 4 and DOB1 subtypes not resolved by the mierotiter plate ~say. A further 16 pairs were found to be rmsmatchod. We have now replaced class 11 serology by the HLA-DR oligotyping microtiter plate assay for pre-transplant histocompatibility typing. It resulted in significant cost reduction, allowing to focus further testing such as MLC and additional DRB (DR4)/DQB/DPB SSO-oligotyptog to DR-matched donors. Overall the increased efficiency in donor searches in terms of shorter delays and precision in the definition of HLA compatibility should have a beneficial impact on patieot's survival.

IV-68 C. Nevthny-Stickel and E.D. Albert Labor for lmmungenetik. Kmderpoliklinik, LMU Mimchen Penenkolerstr. 8a 8000 Mtinchen 2 NONRADIOACTIVE HLA CLASS H TYPING IN A MICRO'HI'ER PLATE FORMAT USING DIGOXIGENIN-LABELED AMPLIFIED DNA AND BIOTI~LABELED OLIGONUCLEOTIDE PROBES We here describe a new nonradioactive rmcrotiter plate assay for the analysis of geneuc variations at the DNA level. The method combines hybridization of oligonucleotide~ with PCR-amplified DNA in the fluid phase with machine-readable detection in the solid phase. C_mnomicDNA is labeled with dagoxagemn during PCR using a nucleotide max contalrtmg DIG- 11-2',3 '-deoxy-uridthe-5-triphosphate (DIG-11-dUTP). The DIG-labeled amplified DNA is hybridized in solution with an oligonucleotide which is labeled with one biotin at its 3'-end using biotin-16-2'.3'-dideoxy-uridthe-5'-triphosphate(BIO-16UTP) catalyzed by DNA deoxynucleotidylexotransferase(TdT). The hybridized complex is immobilized in a steptavidin (SA)-coated macrotiter plate via the biotin and detection of the digoxigenin is performed using .and-chgoxagenin horseradish peroxidase, lab fragments (anti-DIG-POD). and the colonmetric substrate ABTS This method results in highly specific and sensitive hybndization signals and. with the ohgonucleotides chosen, allows the typing of DRI-DR10. The ume required for dus procedure is 2 hours for PCR amplification (could be shorter} and 3 hours for hybridtzauon and detection. The major advantages are I. The relauve safety of oligonucleotide hybridization combined with a fast procedure 2. Direct machine readability for objective evaluation and data processing of test results 3. The method is versatile for application in different oligotypmg systems.