Francesca Drago

Blood group genotyping for Jka/Jkb, Fya/Fyb, S/s, K/k, Kpa/Kpb, Jsa/Jsb, Coa/Cob, and Lua/Lub with microarray beads

BACKGROUND: Traditionally, blood group typing has been performed with serologic techniques, the classical method being the hemagglutination test. Serotyping, however, may present important limitations such as scarce availability of rare antisera, typing of recently transfused patients, and those with a positive direct antiglobulin test. Consequently, serologic tests are being complemented with molecular methods. The aim of this study was to develop a low‐cost, high‐throughput method for large‐scale genotyping of red blood cells (RBCs).

Microarray Beads for Identifying Blood Group Single Nucleotide Polymorphisms

We have developed a high-throughput system for single nucleotide polymorphism (SNP) genotyping of alleles of diverse blood group systems exploiting Luminex technology. The method uses specific oligonucleotide probes coupled to a specific array of fluorescent microspheres and is designed for typing Jk^a/Jk^b , Fy^a/Fy^b , S/s, K/k, Kp^a/Kp^b , Js^a/Js^b , Co^a/Co^b and Lu^a/Lu^b alleles. Briefly, two multiplex PCR reactions (PCR I and PCR II) according to the laboratory specific needs are set up. PCR I amplifies the alleles tested routinely, namely Jk^a/Jk^b , Fy^a/Fy^b , S/s, and K/k. PCR II amplifies those alleles that are typed less frequently. Biotinylated PCR products are hybridized in a single multiplex assay with the corresponding probe mixture. After incubation with R-phycoerythrinconjugated streptavidin, the emitted fluorescence is analyzed with Luminex 100. So far, we have typed more than 2,000 subjects, 493 of whom with multiplex assay, and there have been no discrepancies with the serology results other than null and/or weak phenotypes. The cost of consumables and reagents for typing a single biallelic pair per sample is less than EUR 3.–, not including DNA extraction costs. The capability to perform multiplexed reactions makes the method markedly suitable for mass screening of red blood cell alleles. This genotyping approach represents an important tool in transfusion medicine.

Genotyping of the Kidd blood group with allele-specific oligodeoxynucleotides coupled to fluorescent microspheres.

Dear Sir The Kidd blood group system is clinically important in transfusion medicine because allosensitization to Kidd is responsible for at least 30% of delayed haemolytic transfusion reactions. The protein carrying the antigen for the Kidd blood group is a product of a single gene named JK or SLC14A1 LOHICH is located on chromosome 18q12–q21 (Olivès et al., 1995; Lucien et al., 1998). In Caucasians, the common alleles JK*A and JK*B have a frequency of 51 and 49%, respectively, and define the three phenotypes Jk(aþb–), Jk(a–bþ) and Jk(aþbþ). JK null alleles derived from several genetic alterations have also been described (Irshaid et al., 2000; Sidoux-Walter et al., 2000; Lucien et al., 2002). Serological determination of erythrocyte antigens is simple for most situations, but it may be unreliable in multitransfused subjects and in cases of haemolytic disease of the newborn. Furthermore, as fresh and viable red cells are needed, there may be limits to large-scale testing. In response to these problems, over the past 10 years, DNA-based methods have been described for blood group genotyping (Rozman et al., 2000; Castilho et al., 2002; Reid, 2003). We have exploited Luminex technology (Colinas et al., 2000; Dunbar & Jacobson, 2000) to develop a high-throughput method for single-nucleotide polymorphism (SNP) genotyping of JK*A and JK*B, with the ultimate aim of extending this methodology to large-scale DNA typing of blood group systems. These two alleles are clearly distinguishable by a specific point mutation where the presence of a guanine or adenosine leads to an amino acid substitution from Asp to Asn, respectively. Luminex technology utilizes a mixture of oligonucleotide probes covalently attached to fluorescent microspheres, enabling the creation of an oligonucleotide hybridization assay for a polymerase chain reaction (PCR) product. The PCR-amplified target DNA is biotinylated and streptavidin – phycoerythrin (SAPE) is used as the reporter molecule. The fluorescence emission can be resolved by the Luminex 100 instrument (Luminex Corporation, Austin, TX, USA). A total of 250 blood samples were obtained from volunteer blood donors selected according to their Kidd phenotype. Genomic DNA was isolated using the QIAamp Blood Kit (QIAGEN, Hilden, Germany). For PCR amplification of JK exon 9, the following primers were used: JK-781 5 ́ -CATGCTGCCATA GGATCAT and JK-943 5 ́ -GAGCCAGGAGG TGGGTTTGC (Irshaid et al., 1998). The upstream primer was labelled at the 5 ́ -terminus with biotin. We used oligodeoxynucleotides with a 5 ́ -aminomodifier linked with a C12 carbon chain arm for covalent coupling to carboxylated microspheres. The probe sequences (GenBank accession number AF046026) were as follows: JK*A 5 ́ -AGTAGATG TCCTCAAATG; JK*B 5 ́ -AGTAGATGTTC TCAAATG; positive control (PC) 5 ́ -AGGAAGCC AAGATCTCAA (complementary to a sequence of intron 8 of Kidd gene); and non-sense (NS) 5 ́ -CGTGGATTTCTTCAGAGG. The probes were coupled to the carboxylated microspheres in separate reactions according to a protocol described elsewhere (Colinas et al., 2000; Dunbar & Jacobson, 2000). Hybridization was performed in tetramethylammonium chloride (TMAC) 1.5 buffer (4.5 mol L 1 TMAC), 0.15% sodium dodecyl sulfate, 75 mmol L 1 Tris–HCl (pH 8.0) and 6 mmol L 1 ethylenediaminetetraacetic acid (EDTA) (pH 8.0) at 45 ̊C for 15 min. Afterwards, 100 mL of 6 Saline, Sodium Phosphate, Edta-Triton X-100 (SSPET) (0.9 mol L 1 NaCl, 60 mmol L 1 NaH2PO4, 6 mmol L 1 EDTA and 0.005% Triton X-100) were added. The beads were labelled with freshly made 1 SAPE solution (0.5 mg mL ) in 1 TMAC at 45 ̊C. The samples were then analysed with the Luminex 100 instrument. To test whether our system was suitable for JK*A/ JK*B genotyping, a panel of 50 Jk(aþb–), Correspondence: Francesca Poli, Dipartimento Trasfusionale e di Riferimento per i Trapianti di Organi e Tessuti, Ospedale Maggiore Policlinico, Mangiagalli, Regina Elena, Fondazione IRCCS di Natura Pubblica, via Francesco Sforza 35, 20122 Milan, Italy. Tel.: þ39 0255034239; fax: þ39 0255012573; e-mail: france.poli@policlinico.mi.it Transfusion Medicine, 2005, 15, 499–501 doi: 10.1111/j.1365-3148.2005.00632.x

De Novo Donor-Specific HLA Antibodies Developing Early or Late after Transplant Are Associated with the Same Risk of Graft Damage and Loss in Nonsensitized Kidney Recipients

De novo posttransplant donor-specific HLA-antibody (dnDSA) detection is now recognized as a tool to identify patients at risk for antibody-mediated rejection (AMR) and graft loss. It is still unclear whether the time interval from transplant to DSA occurrence influences graft damage. Utilizing sera collected longitudinally, we evaluated 114 consecutive primary pediatric kidney recipients grafted between 2002 and 2013 for dnDSA occurrence by Luminex platform. dnDSAs occurred in 39 patients at a median time of 24.6 months. In 15 patients, dnDSAs developed within 1 year (early-onset group), while the other 24 seroconverted after the first posttransplant year (late-onset group). The two groups were comparable when considering patient- and transplant-related factors, as well as DSA biological properties, including C1q and C3d complement-binding ability. Only recipient age at transplant significantly differed in the two cohorts, with younger patients showing earlier dnDSA development. Late AMR was diagnosed in 47% of the early group and in 58% of the late group. Graft loss occurred in 3/15 (20%) and 4/24 (17%) patients in early- and late-onset groups, respectively (p = ns). In our pediatric kidney recipients, dnDSAs predict AMR and graft loss irrespective of the time elapsed between transplantation and antibody occurrence.

The allocation of pancreas allografts on donor age and duration of intensive care unit stay: the experience of the North Italy Transplant program.

Starting in 2011, the North Italy Transplant program (NITp) has based on the allocation of pancreas allografts on donor age and duration of intensive care unit (ICU) stay, but not on donor weight or BMI. We analyzed the detailed allocation protocols of all NITp pancreas donors (2011–2012; n = 433). Outcome measures included donor characteristics and pancreas loss reasons during the allocation process. Twenty‐three percent of the 433 pancreases offered for allocation were transplanted. Younger age, shorter ICU stay, traumatic brain death, and higher eGFR were predictors of pancreas transplant, either as vascularized organ or as islets. Among pancreas allografts offered to vascularized organ programs, 35% were indeed transplanted, and younger donor age was the only predictor of transplant. The most common reasons for pancreas withdrawal from the allocation process were donor‐related factors. Among pancreas offered to islet programs, 48% were processed, but only 14.2% were indeed transplanted, with unsuccessful isolation being the most common reason for pancreas loss. Younger donor age and higher BMI were predictors of islet allograft transplant. The current allocation strategy has allowed an equal distribution of pancreas allografts between programs for either vascularized organ or islet transplant. The high rate of discarded organs remained an unresolved issue.

A Microsphere-Based Suspension Array for Blood Group Molecular Typing: An Update

Background: In a previous publication we described a method for Jk^a/Jk^b, Fy^a/Fy^b, S/s, K/k, Kp^a/Kp^b, Js^a/Js^b, Co^a/ Co^b, and Lu^a/Lu^b genotyping based on a microsphere suspension array. Here, an improved version of the assay is presented. Methods: Two multiplex polymerase chain reactions (PCR) were developed: one for amplification of samples routinely tested and the other for those systems that are tested less frequently. Each biotinylated PCR product is hybridized in a single multiplex assay. A total of 2,020 samples were analyzed, and the genotypes were compared to the blood group phenotypes. Results: There have been no discrepancies with the serology results other than null and/or weak phenotypes. Conclusion: In its present form, the method presented here has the capacity to genotype hundreds of a samples in few hours with a high concordance rate with serology.

Use of IgM monoclonal reagents licensed for tube tests in column agglutination technology

BACKGROUND: Red cell (RBC) phenotyping using column agglutination technology (CAT) is currently limited by the reagents formulated in the system. To overcome this limitation, it was investigated whether monoclonal IgM reagents licensed for use with tube tests produced valid results with CAT. STUDY DESIGN AND METHODS: Commercial CAT, does not contain antisera, was used to evaluate Procedures A (40 microL of reagent and 10 microL of 4% RBCs) and B (50 microL of reagent and 50 microL of 0.8% RBCs) with or without incubation at room temperature. In Study 1, reagents were tested to determine whether potentiators inhibit the passage of antigen‐negative RBCs through the column. In Study 2, CAT sensitivity was measured by the use of potency titrations to define a procedure for each reagent that matched or exceeded that of the tube method. In Study 3, the specificity of each reagent was determined in parallel with the CAT and tube tests. Typing of 1644 samples was performed. RESULTS: Study 1: Free passage was obtained with all reagents. Study 2: Immediate‐spin methods using CAT produced the same results as the tube method. Study 3: With 8048 comparisons made, discrepant results were found in 32 transfused patients and in 6 cord blood samples, mainly with Lewis reagents. With comparison of CAT and the standard tube method, complete agreement was obtained with Kell reagents, 99.9‐percent agreement with Kidd reagents, and 98.9‐percent and 99.4‐percent agreement with Lewis reagents. CONCLUSION: Most examined reagents seem suitable for use with CAT.