DNA was extracted from peripheral blood using Qiagen kits (QIAamp DNA Mini Kit®; Qiagen, Valencia, CA, USA). HLA A, B, C, DRB1, DQA1 and DQB1 typing was performed using polymerase chain reaction and amplification using sequence-specific oligonucleotide contained in LABType Kits® (One Lambda Inc., Canoga Park, CA, USA).
Labtype kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
LABType kits are a suite of laboratory products designed for comprehensive human leukocyte antigen (HLA) typing. These kits utilize molecular techniques to accurately determine HLA allele profiles from DNA samples, providing essential data for applications such as organ transplantation, disease association studies, and population genetics research.
Automatically generated - may contain errors
Lab products found in correlation
Qiaamp dna mini kit, by Qiagen (3 mentions)
Hla fusion software, by Thermo Fisher Scientific (2 mentions)
Cellquest pro software, by BD (1 mentions)
Dnaeasy blood and tissue kit, by Qiagen (1 mentions)
Miniseq, by Illumina (1 mentions)
Labscan 3d instrument, by DiaSorin (1 mentions)
Fusion software, by Thermo Fisher Scientific (1 mentions)
Geneamp pcr system 9700 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Labscan 100, by Thermo Fisher Scientific (1 mentions)
Alltype kit, by Thermo Fisher Scientific (1 mentions)
Magna pure nucleic acid isolation kit, by Roche (1 mentions)
Magna pure compact, by Roche (1 mentions)
10 protocols using labtype kit
1
HLA Typing from Blood Samples
2
HLA Typing and Antibody Detection for Heart Transplant
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Patients and donors were molecularly HLA typed by a site‐specific primer (SSP)‐PCR using Olerup SSP® kits (Olerup SSP AB, Saltsjöbaden, Sweden) or by reverse sequence‐specific oligonucleotide‐PCR using LABType™ kits (One Lambda, Inc.) as described by the manufacturers.
For heart transplant recipients, the levels of panel‐reactive HLA class I and II Antibodies (PRAs) in the pre‐transplant sera of patients were determined by flow cytometric analysis using the FlowPRA® test according to the manufacturer's instructions (One Lambda, Inc.). The samples were acquired on a fluorescence activated cell sorting flow cytometer and analysed using the CellQuestPro software (BD Biosciences, San Jose, CA, USA). More recently, HLA antibody detection and specificity‐determination were performed using the Luminex® bead‐based LABScreen™ Mixed and Single Antigen assays, respectively (One Lambda, Inc.).
A prospective complement‐dependent cytotoxicity crossmatch (CDC XM) was performed on patients with pre‐transplant HLA antibodies as previously described.17 Transplantation was done if the CDC XM was negative. Patients without HLA antibodies were tested retrospectively in a T‐ and B‐cell flow cytometric XM.18
For heart transplant recipients, the levels of panel‐reactive HLA class I and II Antibodies (PRAs) in the pre‐transplant sera of patients were determined by flow cytometric analysis using the FlowPRA® test according to the manufacturer's instructions (One Lambda, Inc.). The samples were acquired on a fluorescence activated cell sorting flow cytometer and analysed using the CellQuestPro software (BD Biosciences, San Jose, CA, USA). More recently, HLA antibody detection and specificity‐determination were performed using the Luminex® bead‐based LABScreen™ Mixed and Single Antigen assays, respectively (One Lambda, Inc.).
A prospective complement‐dependent cytotoxicity crossmatch (CDC XM) was performed on patients with pre‐transplant HLA antibodies as previously described.
3
Ultra-resolution HLA Genotyping Protocol
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The DNA for HLA genotyping was processed via the workflows of a European Federation for Immunogenetics (EFI)-accredited Clinical Histocompatibility Laboratory. Low-resolution typing of the HLA-A, HLA-B and HLA-C genes was achieved with LABType kits (One Lambda), which rely on reaction patterns observed when sequence-specific DNA probes immobilized on fluorescent X-MAP polystyrene beads (Luminex) hybridize to biotin-labeled multiplexed gene-specific PCR amplicons. The hybrids were detected with a Liquichip 200 fluorimeter (Qiagen) and HLA allele assignment was performed using HLA Fusion software (One Lambda). Ultra-high-resolution typing of HLA-A, HLA-B and HLA-C was achieved with an ‘in-house’ third-generation sequencing pipeline using Single Molecule, Real-Time (SMRT) DNA sequencing technology (Pacific Biosciences) as previously described (Turner et al., 2018 (link)). Typing results are detailed in Table 3 and Table S1 . Details of the World Health Organization (WHO) nomenclature for factors of the HLA system, and Bw6 epitope determination can be found at www.hla.alleles.org .
4
HLA Typing from Blood DNA
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DNA was extracted from peripheral blood using Qiagen kits (QIAamp DNA Mini Kit, Qiagen, Inc, Valencia, CA). The HLA A, B, C and DRB1 typing was performed using PCR and amplification using Sequence-Specific Oligonucleotide (SSP) contained in LABType kits (One Lambda, Inc, Canoga Park, CA).
5
HLA Typing from Blood DNA
6
HLA Typing by NGS and SSO
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For DNA extraction the DNA easy blood and tissue kit from Quiagen was used. HLA typing was performed using next generation sequencing (NGS) with the NGSgo-AmpX v2 HLA kits (GenDx, Utrecht, Netherlands), and sequenced on an Illumina MiniSeq (Illumina, San Diego, CA). Sequence data were analyzed with NGSengine (GenDx, Utrecht, Netherlands). For samples with low DNA amount, HLA typing was also performed using sequence specific oligomers (SSO) with the LABType kits (One Lambda, Canoga Park, CA). The bead-based analysis was run on a LABScan 3D instrument (Luminex, Austin, TX) and analyzed using the Fusion Software (One Lambda, Canoga Park, CA). All assays were performed according to the manufacturer’s recommendations.
7
HLA-B and MICA Typing by PCR-SSO and Luminex
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To perform the HLA-B and MICA typing, about 5 mL of blood was collected by venipuncture in vacuum tubes (Vacutainer, Becton and Dickson, Oxford, UK) containing ethylene diamine tetraacetic acid (EDTA) as anticoagulant. Then, we extracted the genomic DNA by the separation-column method, using the Biopur kit for DNA extraction (Biometrix, Curitiba, Paraná, Brazil), following the manufacturer's protocol. After adjusting the DNA concentration, obtained by the optical-density method, we amplified the DNA using polymerase chain reaction-sequence specific primers (PCR-SSO) combined with Luminex technology. The genomic DNA was amplified using biotinylated sequence-specific primers for HLA-B and MICA in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA), followed by hybridization with complementary probes for DNA, conjugated with microspheres (beads) labeled with different fluorochromes to identify complementary sequences of the amplified DNA, using the LABType kit (One Lambda, Inc., Canoga Park, CA, USA), following the manufacturer's protocol. After hybridization, the results were read using the flow cytometry platform LABScan™100 (One Lambda, Inc.), followed by analysis using the program HLA Fusion version 2.0 (One Lambda, Inc.). The results showed low-medium resolution.
8
High-Resolution HLA Typing Protocol
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High‐resolution typing of HLA, A, B, C, DRB1, DRB3/4/5, DQB1, DQA1, DPA1, and DPB1 was determined by sequence‐specific oligonucleotide probe method (LabType kit, One Lambda, West Hills, CA) and/or by next generation sequencing (AllType NGS kit, One Lambda).
9
High-Resolution HLA Typing by NGS and Sanger
10
HLA Typing for Celiac Disease Susceptibility
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DNA was isolated from the whole blood using the Magna Pure compact instrument (Roche Diagnostics, Mannheim, Germany) and the MagNa Pure Nucleic Acid Isolation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. HLA-Typing for celiac disease susceptibility genes was performed for the 195 randomly selected specimens using Sequence Specific Oligonucleotide (SSO) method. In summary, the SSO method is a DNA-based tissue typing technique using polymerase chain reaction to amplify the target DNA. The amplified product was hybridized with DQA1-specific and DQB1-specific nucleotide probes bound to fluorescently labeled beads that identified alleles encoded in the DNA sample. The process was carried out using Luminex technology in the Lab Type Kit (One lambda®, Canoga Park, USA). Reactivity patterns were interpreted with HLA Fusion software (One lambda®, Canoga Park, USA) to identify the alleles present. Further details of the procedure are available at http://www.onelambda.com .
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