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BACKGROUND: The Luminex Single-Antigen Beads (LSA) assay allows an accurate detection and characterization of preexisting donor-specific antibodies (DSA) in kidney transplant candidates. But the ability of LSA to detect quite low levels of antibodies makes it hard to correctly predict crossmatch results in donor selection. In this study we retrospectively analyzed the accuracy of our virtual crossmatch (v-XM) protocol, which was used for selection of potential kidney transplant recipients, in predicting the results of actual crossmatch (a-XM) in cadaver-donor renal transplantation. We also investigated correlation between negative a-XM results and strength/specificity of preformed DSA. METHODS: The correlation between negative v-XMs and a-XMs performed in 2007-2012 at the Regional Transplant Center of the Lazio Region, Italy, was analyzed. In carrying out v-XM, the donor HLA molecules against which patients showed LSA-detected DSA with normalized mean fluorescence intensity (MFI)>=5,000 were considered to be "unacceptable DSA," and LSA-DSA showing MFI

TIPIZZAZIONE HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1, -DPB1 DEL POTENZIALE DONATORE D'ORGANO MEDIANTE TECNICA RT PCR-SSP. Giuseppina Ozzella1,2, Elvira Poggi1,2, Antonio G. Bianculli1, Damiano Colasante1, Maria R. Fazio1, Andrea Giaffreda1, Annarita Manfreda1, Silvia Sinopoli1, Lucia Spano1, Antonina Piazza1,2. 1 Centro Regionale Trapianti - Lazio, Roma 2 C.N.R. IFT UOS di Roma S. Camillo, Roma Introduzione. Pazienti in lista d'attesa per trapianto d'organo, sensibilizzati da pregressi eventi immunizzanti, quali trasfusioni, gravidanze o trapianti, possono sviluppare ampi patterns anticorpali, specifici per molecole HLA di classe I e II. Le nuove tecniche citofluorimetriche in fase solida, attualmente utilizzate per la caratterizzazione di allo-anticorpi anti-HLA, evidenziano infatti che i pazienti sensibilizzati possono produrre anticorpi anti-HLA specifici non solo per le molecole HLA-A,-B e DRB1, attualmente considerate nel matching donatore/ricevente, ma anche per i loci HLA-C, -DRB3/4/5, -DQA1, -DQB1, -DPA1 e DPB1, sottolineando così, anche in accordo con le Linee-Guida proposte dalla Società Italiana di Immunogenetica e Biologia dei Trapianti (AIBT), la necessità di estendere la tipizzazione a tutti i loci HLA del potenziale donatore. L'impiego della nuova tecnica di biologia molecolare Real Time PCR SSP permette di eseguire, in tempi molto brevi, la tipizzazione HLA del potenziale donatore d'organi per tutti i loci HLA di classe I e II consentendo così, attraverso un più accurato virtual crossmatch pre-trapianto, di selezionare il ricevente più idoneo al trapianto e, quindi, di valutare il reale rischio immunologico nei riceventi selezionati. Materiali e metodi. Il protocollo attualmente in uso presso il Laboratorio di Tipizzazione Tissutale ed Immunologia dei Trapianti del CRT-Lazio per la tipizzazione dei donatori d'organo prevede l'utilizzo della tecnica di PCR-SSP a bassa risoluzione per i soli loci -A, -B, -C, -DRB1 e -DQB1. Al fine di estendere la tipizzazione anche agli altri loci HLA (-DRB3/4/5, -DQA1, -DQB1, -DPA1 e DPB1), finora tipizzati pre-trapianto solamente in caso di selezione di potenziali riceventi con anticorpi specifici per dette molecole, abbiamo eseguito la validazione della tecnica di tipizzazione genomica RT PCR-SSP con l'impiego di prodotti della linea LinkSeq (Linkage Bioscience Inc., Voden Medical Instrument Spa) e dello strumento QuantStudio 5 (Thermo Fisher Scientific), con software applicativo SureTyperTM. Questa tecnica combina l'amplificazione con primers allele/gruppo specifici con una rilevazione diretta dei prodotti amplificati attraverso l'uso del colorante SYBR Green, in grado di legare il DNA a doppia elica. Lo strumento di RT PCR misura, in tempo reale, la fluorescenza emessa durante la fase esponenziale dell'amplificazione e registra il decadimento della fluorescenza nel ciclo finale di dissociazione. Il Software dedicato processa i dati di dissociazione, generando, per ogni pozzetto della piastra, una curva di melting che è esaminata come deriva negativa dei valori di fluorescenza (-dF) rispetto ad un range di temperatura (dT), rappresentata, in un sistema di assi cartesiani (-dF/dT), come "picchi di melting", definiti positivi/negativi rispetto a valori attesi di reazioni note. L'analisi dei patterns di reazioni permette l'identificazione degli alleli HLA presenti nel campione. Mediante tale tecnica sono stati tipizzati retrospettivamente, o in contemporanea alla tecnica in uso, 10 potenziali donatori d'organo e le tipizzazioni ottenute sono state comparate con le tipizzazioni eseguite mediante tecnica di PCR-SSP a bassa risoluzione. Risultati. L'analisi dei risultati ha evidenziato che la tecnica RT PCR-SSP (Tabella 1) è in grado di fornire, in tempi brevi (circa 90 minuti), la tipizzazione di 11 loci HLA (-A, -B, -C, -DRB1, -DRB3,4,5, -DQA1, Tab. 1RT PCR-SSP N. TipABCDRB1DQA1DQB1DRB3/4/5DPA1DPB1 10484*01*30*42*53*04*17*11*16*01*05*03*053*01/035*01/02*01*02*02:01*04:01 12862*24*24*18*35*04*12*11-*05-*03-3*02-*01 *04:02 12866*02*24*08*35*04*07*07*11*02:01*05:01*02*033*024*01*01:03*02:01*03:01*63:01 13363*03*24*35*51*04*15*04*11*03*05*03-3*024*01*01 *03:01*04:01 14177*02*26*49*51*01*07*01-*01-*05---*01*02*03:01*10:01 14184*03*30*35*44*02*16*03*13:02*01*04:01*04*063*013*02*02 *01:01*10:01 14195*03*26*38*51*12*15*13*16*01-*05*063*01:015*02:02*02:01 *05:01*13:01 14226*01*02*35*44*04*16*04*11*03*05*03*033*024*01*01 *02:01*04:01 14227*30*30*13*51*06*15*03*07*02:01*05*02 3*014*01*01 *04:01*04:02P 14256*03*26*35*55*03*04*10*11*01*05*03*053*02-*01*02*04:01*14:01 -DQB1, DPA e DPB) ad un livello intermedio di risoluzione e consente di eliminare diverse ambiguità alleliche delle tecniche genomiche di PCR-SSP a bassa risoluzione (Tabella 2). Da un punto di vista tecnico, con l'uso di tale metodica, sono completamente eliminati numerosi passaggi manuali, come la preparazione del gel di agarosio e la manipolazione del bromuro di etidio. Tab. 2PCR-SSP N. TipABCDRB1DQA1DQB1DRB3/4/5DPA1DPB1 10484*01*30*42*53*04*17*11*16*01*05*03*05-- 12862*24*24*18*35*04*12*11-*05:05-*03-DRB3- *04:02 12866*02*24*08*35*04*07*07*11--*02*03DRB3DRB4 *03:01*63:01 13363*03*24*35*51*04*15*04*11*03:03*05:05*03-DRB3DRB4 *03:01*04:01 14177*02*26*49*51*01*07*01---*05--- 14184*03*30*35*44*02*16*03*13:01/02/15--*04*06DRB3- 14195*03*26*38*51*12*15*13*16--*05*06DRB3DRB5 14226*01*02*35*44*04*16*04*11--*03*03DRB3DRB4 14227*30*30*13*51*06*15*03*07--*02-DRB3DRB4 14256*03*26*35*55*03*04*10*11--*03*05DRB3- La tipizzazione dei potenziali donatori mediante tale tecnica avrebbe inoltre, in un caso (10%), evitato di eseguire una tipizzazione aggiuntiva del locus DRB1 a causa di una ambiguità ottenuta con la tecnica di PCR-SSP a bassa risoluzione, dovuta ad alleli "common" e "well documented" e, in 4 casi (40%), avrebbe evitato da una parte l'esecuzione di tipizzazioni aggiuntive dei loci DQA1 e DPB1 e, dall'altra, avrebbe permesso l'esclusione di due pazienti dall'esecuzione dei crossmatch pre-trapianto, poiché tali pazienti avrebbero dato virtual crossmatch positivo a causa di anticorpi donatore-specifici con intensità di fluorescenza elevata (MFI >5000). Conclusioni. La nuova tecnica RT PCR-SSP ha mostrato di essere uno strumento idoneo ad ottimizzare il matching donatore/ricevente, poiché, fornendo una tipizzazione HLA estesa a tutti i loci, con una sensibile riduzione del numero di ambiguità, consente l'esecuzione di un più accurato virtual crossmatch, utilizzato al fine di selezionare i riceventi più idonei per un donatore. Permette, quindi, una più attenta valutazione del "rischio immunologico" del potenziale ricevente di trapianto d'organo e, inoltre, risulta idonea in termini di sostenibilità economica nonché di compatibilità con l'urgenza di esecuzione della tipizzazione HLA del potenziale donatore, fornendo i risultati in tempi più rapidi rispetto a quelli che sono necessari utilizzando metodiche di PCR-SSP a bassa risoluzione.

We describe an additional HLA-Cw*02 variant, HLA-Cw*0208, which has been identified in a renal transplant recipient of Caucasian origin (Italy). After performing preliminary serological typing, we analyzed exons 2 and 3 of the HLA-C locus polymorphism by cloning the amplified DNA and using a sequence-based typing method. The new allele differs from Cw*020202 by one nucleotide substitution at nucleotide 61 (G-->A) of exon 2, which translates to a difference of one amino acid at residue 21 (His-->Arg) of the HLA-C heavy chain. We propose that Cw*0208 was generated by a random point mutation in codon 21 from the Cw*020202 allele, or through gene conversion of Cw*020202 with another allele, probably the Cw*1205 and Cw*1602 alleles.

Aim: All preformed HLA antibodies have a deleterious impact on kidney graft outcome. Alpha and beta chains of DQ and DP molecules can elicit a humoral immune response. Methods: In 191 HLA class II-positive renal transplant recipients we investigated the incidence of DQ and DP alloantibodies by Single antigen beads coated with 29 DQA1/DQB1 and 24 DPA1/DPB1 heterodimers (One Lambda Inc). We also analyzed the putative epitopes for antibody formation by analysis of amino acid sequences of molecules corresponding to detected specificities. All patients were DQA1/DQB1 and/or DPA1/ DPB1 typed by SSP assays. Results: Anti-DQ antibodies were detected in 126 patients. Ninety-four (75%) had had a previous transplant; 14 of these only developed anti-DQ antibodies. Seventy-one percent of patients had anti-DQB1 antibodies, 2% had anti-DQA1 and the remaining 29% had both. As for the epitopes that might determine antibody-patterns development, 28 epitopes were identified (11 for DQA1 and 17 for DQB1); 7 epitopes had not been reported yet (DQA1: 41K/130A0103; 50L02, 03; 52R03, 04, 05, 06; 69T04, 06; 75I01, 02, 03, 04, 06; 160D0302, 0303. DQB1: 87Y05, 0604/05/07/09). Anti-DP antibodies were detected in 64 patients. Fifty-one (80%) had had a previous transplant; 6 of these only developed anti-DP antibodies. Eightyone percent of patients had anti-DPB1 antibodies, 3% had anti-DPA1 and the remaining 16% had both. As for the putative epitopes, we identified 1 epitope characteristic of DPA1 and 10 epitopes characteristic of DPB1 molecules. Nine DPB1 epitopes were located in the 6 hypervariable regions of exon 2 of the DPB1 alleles, while one epitope (96R:*02, 04, 17, 23, 28) was outside of these regions. Conclusions: These data show that both alpha and beta chain epitopes of DQ and DP molecules are immunogenic and indicate the importance of epitope-identification studies.

Incidence and epitope specificity of HLA DQA1 and DQB1 antibodies in renal transplant recipients Antonina Piazza1, Giuseppina Ozzella1, Elvira Poggi1, Daniela Caputo2, Rosa Cremona2, Valentina Imbroglini2, Domenico Adorno1 1C.N.R. - Istituto per i Trapianti d'Organo, Rome, Italy, 2Centro Regionale Trapianti - Lazio, Rome, Italy Since epitopes of HLA molecules represent the binding site of alloantibodies, the exact characterization of HLA antibodies has an important for the management of sensitized renal transplant candidates. The recent development of assays using Single Antigen beads coated with DQ heterodimers (DQA1 and DQB1) permit to distinguish DQA1 from DQB1 alloantibodies. Besides, analyzing the beads' reaction patterns in relation to amino acid sequences of antibody-reactive alleles it is possible to identify DQA1 and DQB1 sensitizing epitopes. In 173 renal transplant candidates showing production of HLA class II antibodies we characterized anti-DQ antibodies using HLA class II Single Antigen beads containing 12 DQA1 and 14 DQB1 alleles variously combined each other. One hundred and four (60%) of the HLA class II positive patients produced anti-DQ antibodies; 88 of the 104 recipients had had a previous transplant. Sixty-eight patients had only anti-DQB1 antibodies, 2 had only anti-DQA1 and the remaining 34 had both anti-DQB1 and anti-DQA1. Anti-DQ positive patients were typed for DQA1 and DQB1 alleles by PCR-SSP technique. Correlating the reaction pattern of each antibody to the amino acid sequences of DQA1/DQB1 alleles, we could identify 9 epitopes characteristic of DQA1 molecules and 14 epitopes characteristic of DQB1. Seven (30%) of these 23 epitopes had not been reported yet; 6 were DQA1-epitopes (41K/130A = DQA1*0103; 160D = DQA1*0302, 0303; 75S/107I/161E/163S/175K = DQA1*05; 50L = DQA1*02, 03; 69T = DQA1*04, 06; 75I = DQA1*01, 02, 03, 04, 06) and one was a DQB1-epitope (87Y = DQB1*05, 0604, 0605, 0607, 0609, . . .). This study, carried out on a large number of HLA-DQA1/DQB1 sensitized patients, confirms the great immunogenicity of mismatched DQ molecules of a renal transplant. Furthermore the characterization of detected HLADQ antibodies allowed us to identify seven unreported epitopes of the DQA1/DQB1 molecules. These findings may be useful in increasing our knowledge of HLA epitope repertoire which can lead to an accurate analysis of high panel reactive antibodies.

The aim of this study was to provide genetic and anthropological information on the Chaouya (CH), an Arabic-speaking population living in West Morocco, Atlantic coast (Settat). In 98 unrelated healthy CH volunteers, we first investigated the human leukocyte antigen (HLA) class I and II allele polymorphisms using a sequence-based typing method and examined haplotypes and relatedness of this group to other African and Mediterranean populations. The study showed the close relatedness with Tunisian population and other North Africans, together with a strong influence of various immigrations, mainly Spaniards, French, and Portuguese, as expected. Nevertheless, analysis of class II allele frequencies (afs) showed that Oromo and Amhara Ethiopian groups cluster together with the Berbers and other North Africans, confirming the relationship between these populations (Afro-Asiatic linguistic group, Hamites). South and sub-Saharan Africans cluster separately at a great distance from CH, except the sub-Saharan Bantu population from Congo Kinshasa, which shows a relatively close genetic relationship ascribable to the effect of a diversifying selection. On the other hand, considering HLA class I afs analyses, it was noteworthy that CH grouped together with sub-Saharans, showing a close genetic distance mainly with Ugandas and Kenians Luo.

Post-transplant development of donor-specific antibodies (DSA) represents a negative prognostic factor in kidney transplantation. This study analysed the impact of posttransplant HLA- and MICA-DSA on kidney graft outcome.

B*3812 is a novel allele identified in our laboratory during the typing procedure for hematopoietic cell transplantation purpose. The name B*3812 has been officially assigned by the WHO Nomenclature Committee in November 2005.

Background Killer cell immunoglobulin-like receptors (KIRs) are a family of inhibitory and activatory receptors that are expressed by most natural killer (NK) cells. The KIR gene family is polymorphic: genomic diversity is achieved through differences in gene content and allelic polymorphism. The number of KIR loci has been reported to vary among individuals, resulting in different KIR haplotypes. In this study we report the genotypic structure of KIRs in 217 unrelated healthy Italian individuals from 22 immunogenetics laboratories, located in the northern, central and southern regions of Italy. Methods Two hundred and seventeen DNA samples were studied by a low resolution PCR-SSP kit designed to identify all KIR genes. Results All 17 KIR genes were observed in the population with different frequencies than other Caucasian and non-Caucasian populations; framework genes KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2 were present in all individuals. Sixty-five different profiles were found in this Italian population study. Haplotype A remains the most prevalent and genotype 1, with a frequency of 28.5%, is the most commonly observed in the Italian population. Conclusion The Italian Caucasian population shows polymorphism of the KIR gene family like other Caucasian and non-Caucasian populations. Although 64 genotypes have been observed, genotype 1 remains the most frequent as already observed in other populations. Such knowledge of the KIR gene distribution in populations is very useful in the study of associations with diseases and in selection of donors for haploidentical bone marrow transplantation.

Sensitivity in detecting low levels of alloantibodies and specificity in revealing the presence of HLA-specific antibodies are the most important clinical features of a PRA screening technique. In order to improve both aspects in revealing a pre-sensitisation status, 297 sera from 221 patients awaiting renal transplantation were screened using Amos-CDCPRA and FlowPRA class I and II beads techniques. Overall concordance was 72.7%. Moreover 48 (16.2%) serum samples showed “false negative” CDCPRA results and 33 (11.1%) sera presented “false positive” CDCPRA results. As regards the percentage of PRA positivity, we highlighted higher mean PRA levels using FlowPRA than CDCPRA technique (66%±26% vs. 37%±25%, p

Messa a punto della tecnica di sequenziamento degli antigeni HLA, utilizzando i primers locus-specifici per gli esoni d'interesse, al fine di identificare il maggior numero di alleli possibili per una migliore tipizzazione dei pazienti in lista di attesa per trapianto di rene.

Contributions presented at the International Symposium BIOTECH RIA '88, "Molecular Probes: Technology and Medical Applications", held in Florence, Italy, April 11-13, 1988