Authors:
X. Lafarge (Bordeaux, FR)
B. Bardy (FR)
G. Bertrand (Rennes, FR)
C. BOUILLE (Poitiers, FR)
A. Cesbron (FR)
V. Dubois (Lyon, FR)
C. FRASSATI (Marseille, FR)
I. Jollet (Poitiers, FR)
D. Masson (La Tronche, FR)
P. Moskovtchenko (Lyon, FR)
A. Parissiadis (Strasbourg, FR)
C. Picard (Marseille, FR)
B. PROUST (Tours, FR)
V. Renac (Rennes, FR)
s. TOURNE (Strasbourg, FR)
A. Walencik (Nantes, FR)
i. desbois (Paris, FR)
This study presents the experience of eight French laboratories that utilized the HOLOTYPE HLA kits (OMIXON) for performing HLA typing. This technique necessitates one dedicated PCR per locus. Several kits exist for different numbers of samples (24-96) and loci to type (2, 5, 7 or 11). Two different PCR programs are necessary for class I and II. Amplicon quantification is recommended by the manufacturer. Appropriately diluted amplicons are pooled for each individual and then fragmented, enzymatically, repaired and tagged with indexes. Indexed fragments are pooled and purified with magnetic beads. A selection of the appropriate fragments is performed based on their size by preparative electrophoresis. Afterwards, the library is quantified by qPCR. After appropriate dilution, the library is sequenced on a MiSeq (ILLUMINA). The interpretation of the sequences obtained is done by the HLATwin software using systematically two algorithms. The reliability of the interpreted results is expressed by the calculation of a number of quality criterias. The validation of the kits was performed in all eight laboratories. The sensitivity/specificity was evaluated on 20 EPT samples. The results obtained with the previous methods used in the laboratories (SBT Celera, SSO reverse Luminex One Lambda + PCRSSP OLERUP) were compared on routine samples, including particular samples (new and null alleles). Results revealed excellent concordance with consensual typings obtained in EPT samples, or with results obtained with the other techniques on routine samples. The 4-6 digit resolution is excellent and new alleles are detected. In addition, quantification of the amplicons is not necessary. The only discrepancies observed in the results obtained, were due to new alleles not detected with the previously used technique. They concerned polymorphisms not explored by the previous technique: positions not tested by SSO probes, or exons not routinely sequenced in SBT.