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Transplant Biology & HLA

Human leukocyte antigen (HLA) is a protein-or marker-found on most cells in humans and is used to match with a donor for organ, bone marrow or cord blood transplants. HLAs correspond to major histocompatibility complex (MHC) proteins in humans with six genes (three from each parent) for class I and six different class II genes. MHC class I genes are called HLA -A, -B, and -C; those for class II genes are called HLA-DP, -DQ, and -DR. All these loci express a high degree of polymorphism and each year many new alleles are discovered decreasing the likelihood of two unrelated individual having identical class I and II proteins. A complete characterization of closely matched alleles between two different individuals help for transplantation rejection. Following an organ transplantation the patient has to be carefully monitored for complications including infection and rejection. Integrated Gulf Biosystems (IGB) offers a whole range of solutions from multiple principals for HLA typing ranging from Serology based matching to using Next Generation Sequencing (NGS) methods, we also offer a range of post-transplant monitoring products.

Solutions from

  • Pre-Transplant Matching
  • Post-Transplant Monitoring

Pre-Transplant Matching

To address the diverse needs of all the HLA labs including basic research to advanced diagnostics labs across the Middle East, IGB has a range of solutions starting from serology to molecular level detection products like Sequence-specific primers (SSP), Sequence-specific oligonucleotides (SSO), sequence-based typing (SBT), Real-Time PCR (qPCR) and Next generation sequencing (NGS).

SerologySSPSSOSBTNGS

Serology

HLA typing using antibodies has remained a gold standard for years for diagnostic purposes or to investigate harmful consequences of transfusion. The process involves the measurement of HLA-specific antibodies (or other platelet-specific antibodies) in the patient with a simple micro lymphocyte toxicity test (MLCT), Where the anti-HLA sera react with corresponding, membrane-bound antigens of human lymphocytes and the addition of rabbit complement leads to structural changes of the cell membrane, so that an indicator dye can penetrate the lymphocytes and tint them (positive reaction). If an antigen-antibody reaction does not occur, the cell membrane remains intact. The cells are unable to absorb the dye (negative reaction).

Product

HISTO TRAY B27 (10)  
HISTO TRAY B27 (20)
HISTO TRAY B27 (50)
HISTO TRAY B27 (10) forte
HISTO TRAY AB 72 (10)
HISTO TRAY AB 120 (5)
HISTO TRAY AB 144 (5)
HISTO TRAY ABC 72 (10)
HISTO TRAY ABC 120 (5)
HISTO TRAY ABC 144 (5)
HISTO TRAY DR 72 (10)
HISTO TRAY Complement ABC 72 (10)

Detailed protocols:

IFU-HISTOTRAY-HLA-Class-I-V3-2016-DE-EN
IFU-HISTOTRAY-HLA-Class-II-V3-2016-DE-EN
MSDS-HISTOTRAY-2014-04-EN

HISTOPREP Isolation of lymphocytesHISTOTRAY pre-dropped anti-HLA seraRabbit complementRead under a fluorescence microscope

HISTOPREP is a separation medium for the isolation of lymphocytes with a density of 1.077 g/ml and are made from a highly cross-linked polymer of sucrose. The solution is ready to use. Featuring physiologic conditions, HISTOPREP ensures a high viability of lymphocytes after separation.

The HISTO TRAY test trays are based on the micro lymphocyte toxicity test (MLCT). In this test, anti-HLA sera react with corresponding, membrane-bound antigens of human lymphocytes. The addition of rabbit complement leads to structural changes of the cell membrane so that an indicator dye can penetrate the lymphocytes and tint them (positive reaction). If an antigen-antibody reaction does not occur, the cell membrane remains intact. The cells are unable to absorb the dye (negative reaction).


SSP

Sequence-specific primers (PCR-SSP)

Sequence-specific primers (PCR-SSP) is a relatively straight forward technique which was developed more than two decades now, where multiple pairs of cis-located allele-specific primers will determine the alleles present in a given DNA sample.

HISTO TYPE SSP Diagnostics

  • The new kit format of HISTO TYPE SSP kits including (Taq polymerase)
  • Achieve reliably clear results with the CE certified and validated Taq Polymerase that is delivered with the kit
  • HISTO MATCH software is provided free of charge for the interpretation and documentation of your results

Detailed protocols:
IFU-HISTOTYPE-V17-2017-EN (pdf)
IFU-HappyTaq-V2-2015_DE-EN-IT (pdf)
IFU-HISTOMATCH-V18-2017-EN (pdf)

DNA Extraction and Quantification

Our solutions for DNA extraction for HLA workflows are as follows:

JetQuick™ Blood and Cell Culture DNA Maxiprep Kit (Invitrogen™)
KingFisher™ Pure DNA Blood Kit (Thermo Scientific™)
DNAzol™ BD Reagent, for isolation of genomic DNA from whole blood (Invitrogen™)
GeneJET Whole Blood Genomic DNA Purification Mini Kit (Thermo Scientific™)
JetFlex™ Genomic DNA Purification Kit (Invitrogen™)
GeneCatcher™ gDNA Automated Blood Kit (Invitrogen™)
PureLink™ Genomic DNA Mini Kit (Invitrogen™)
iPrep™ PureLink™ gDNA Blood Kit (Invitrogen™)
To learn more about Nucleic acid isolation visit our Workflow

PCR amplification

All pre-aliquoted and dried reaction mixtures already contain allele and control-specific primers and nucleotides. located polymorphismsThese are supplied dried down in the reaction vial. Amplification parameters are optimized to a final volume of 10 µl.

Our solutions

70721 HISTO TYPE A low 20 Tests
70731 HISTO TYPE B low 20 Tests
70741 HISTO TYPE C low 20 Tests
70751 HISTO TYPE DR low 20 Tests
70891 HISTO TYPE DQB low 20 Tests
7102 HISTO TYPE ABC 20 Tests
7103 HISTO TYPE DR/DQB 20 Tests
7098 HISTO TYPE ABDR 20 Tests
709010 HISTO TYPE DQB high 10 Tests
7070 HISTO TYPE B27 low 48 Tests
7071 HISTO TYPE B27 low 96 Tests
70715 HISTO TYPE B*57:01 / B*51 20 Tests
70941 HISTO TYPE Celiac Disease 20 Tests
70716 HISTO TYPE Narcolepsy 20 Tests

Gel Electrophoresis

To learn more about Gel Electrophoresis and imaging visit our Workflow

Data Analysis

HISTO Type SSP Module

This view shows the results of the interpretation. The default view shows the possible 2 digit results, along with number of mismatches (MM) required to get the result and the frequency of the alleles found (CWD column). Green indicates a C allele, blue a WD allele and grey a rare allele.


SSO

Sequence-specific oligonucleotide (SSO) Workflow

The HISTO SPOT SSO system is a fully automated in vitro diagnostic test for tissue typing of HLA alleles on a molecular genetic basis and provides medium to higher resolution typing results of the alleles in the CWD 2.0.0 catalog (Mack S.J. et al., 2013). Target DNA is PCR-amplified using biotinylated group-specific primers. A single PCR reaction is used for each HLA locus. The biotinylated PCR product is denatured and allowed to rehybridize to a complementary array of immobilized sequence-specific oligonucleotide (SSO) probes. These probes are either single oligonucleotide probes or a combination of 2 or more individual probes, immobilized in the same spot (Mosaic Probes) which have been designed to improve the identification of cis located polymorphisms.DRB3/4/5

DNA Extraction

Our solutions using Automated and semi-automated Methods of DNA Extraction:

JetQuick™ Blood and Cell Culture DNA Maxiprep Kit (Invitrogen™)
KingFisher™ Pure DNA Blood Kit (Thermo Scientific™)
DNAzol™ BD Reagent, for isolation of genomic DNA from whole blood (Invitrogen™)
GeneJET Whole Blood Genomic DNA Purification Mini Kit (Thermo Scientific™)
JetFlex™ Genomic DNA Purification Kit (Invitrogen™)
GeneCatcher™ gDNA Automated Blood Kit (Invitrogen™)
PureLink™ Genomic DNA Mini Kit (Invitrogen™)
iPrep™ PureLink™ gDNA Blood Kit (Invitrogen™)
To learn more about Nucleic acid isolation visit our Workflow

Locus specific PCR amplification

MR.SPOT® Platform-The Concept

One platform-many different applications (HLA and Blood Typing)

  • Genetic HLA-typing kits (A, B, C, DR, DQ, DP)
  • Genetic blood group typing kits (RHD, KEL, JK, FY, MNS, LU, DO, CO, DI, VEL and more)

Hybridization of amplicons to probes

Locus specific PCR amplification

HISTO SPOT® A 4D 96 Tests 726010
HISTO SPOT® A Xtend 24 Tests 726011
HISTO SPOT® B 4D 96 Tests 726020
HISTO SPOT® B Xtend 24 Tests 726021
HISTO SPOT® C 4D 96 Tests 726030
HISTO SPOT® DRB1 4D 96 Tests 726040
HISTO SPOT® DRB1 Xtend 24 Tests 726041
HISTO SPOT® DRB 3/4/5 24 Tests 726045
HISTO SPOT® DQB1 4D / DQA 196 Tests 726051
HISTO SPOT® DPB1 96 Tests 726060
HISTO SPOT® DPB1 24 Tests 726061
HISTO SPOT® Coeliac Disease 48 Tests 726071
HISTO SPOT® On-call Typing Kit 10 Tests 726070

HISTO SPOT® Locus specific tests

Hybridization Reagents

HISTO SPOT® Conjugate 40 µl 726202
HISTO SPOT® Blocking Buffer 80 ml 726201
HISTO SPOT® TBS Wash Buffer 100 ml 726094
HISTO SPOT® Stringent Wash Buffer 100 ml 726093
HISTO SPOT® Hybridization Buffer 40 ml 726092
HISTO SPOT® Substrate 36 ml 726091
HISTO SPOT® Mastermix A (on request) 1100 µl 726080
HISTO SPOT® Mastermix B (on request) 1100 µl 726081
HISTO SPOT® Mastermix C (on request) 1100 µl 726083
HISTO SPOT® Mastermix DRB1 (on request) 1100 µl 726084
HISTO SPOT® Mastermix DQ (on request) 1100 µl 726082
HISTO SPOT® Mastermix DPB1 (on request) 1100 µl 726086
HISTO SPOT® Mastermix DRB3/4/5 (on request) 330 µl 726204
HISTO SPOT® Mastermix Coeliac Disease (on request) 650 µl 726203
HISTO SPOT® Magnesium Chloride 600 µl 726087

HISTO SPOT® Hybridization reagents

Data Analysis

HISTO SPOT® SSO Module

HISTO SPOT® HLA-Ab Module

  • Patient and sample management
  • Create worklists and test layouts
  • Communication with the MR.SPOT®  processor via network
  • Automatic interpretation and easy editing of HISTO SPOT® SSO, HISTO TYPE SSP and ERY SPOT® SSO results
  • User defined typing reports
  • Database management
  • Different user categories
  • different data import and export formats, including SCORE™ export

SBT

Sanger Sequencing Based Typing (SBT)

As is the case with SSP and SSO, SBT also precedes with a locus specific PCR amplification to generate sequencing templates. The reaction needs DNA (Single stranded), locus specific primers, polymerase enzyme, deoxynucleotide phosphates (dNTPs) and fluorescently labelled di-deoxynucleotide phosphates (ddNTP’s). Once amplified, the fragments are purified and are analyzed using automated high-throughput DNA sequence analyzers and the generated sequenced data is interpreted as peak traces on a chromatogram.

SBTexcellerator

Accelerate your high resolution HLA typing.

SBTexcellerator covers a wide range of optimised reagents for Sanger Sequencing Based Typing (SBT). The product line includes HLA Class I and HLA Class II typing kits, which are fully compatible with a powerful SBTengine® analysis software.

The assay set-up provides an efficient combination of generic and Group Specific Sequencing Primers (GSSPs) to accomplish enhanced ambiguity resolution in one single round of sequencing.

DNA Extraction and Quantification

Our solutions for Automated and semi-automated Methods of DNA Extraction:

JetQuick™ Blood and Cell Culture DNA Maxiprep Kit (Invitrogen™)
KingFisher™ Pure DNA Blood Kit (Thermo Scientific™)
DNAzol™ BD Reagent, for isolation of genomic DNA from whole blood (Invitrogen™)
GeneJET Whole Blood Genomic DNA Purification Mini Kit (Thermo Scientific™)
JetFlex™ Genomic DNA Purification Kit (Invitrogen™)
GeneCatcher™ gDNA Automated Blood Kit (Invitrogen™)
PureLink™ Genomic DNA Mini Kit (Invitrogen™)
iPrep™ PureLink™ gDNA Blood Kit (Invitrogen™)
To learn more about Nucleic acid isolation visit our Workflow

Amplification

The amplification assays with an excellent exon coverage provides a combination of generic and Group Specific Sequencing Primers (GSSPs) to accomplish enhanced ambiguity resolution in one single round of sequencing. There is a huge range of choice provided to amplify the desired locus of interest and select very specific kits.

A B C DPA1 DPB1 DQA1 DQB1 DRB1 DRB3/4/5 G

Some of our solutions for this workflow:

Purification

Once amplified the PCR products should be purified to remove the excess of primers and the unincorporated dntp’s, (to have a clear defined peaks). ExoSAP-IT utilizes two hydrolytic enzymes, Exonuclease I and Shrimp Alkaline Phosphatase to remove unwanted dNTPs and primers from PCR products.

Catalog number Price (SAR)
78200.200.UL 100 reactions
78201.1.ML 500 reactions
78202.4X.1.ML 2000 reactions
78205.10.ML 5000 reactions

Capillary Sequencing

Sequencing products are generated by cycle sequencing then purified to remove fluorescent ddNTP’s. The products of the sequencing reaction are injected into capillaries filled with polymer to perform capillary electrophoresis. More info on Capillary sequencing

Catalog number Price (SAR)
4337454 24 reactions
4337455 100 reactions
4337456 1,000 reactions
4337457 5,000 reactions
4337458 25,000 reactions
BigDye™ Terminator v3.1 Cycle Sequencing Kit
Seq Studio Genetic Analyzer
(4 Capillary)
3500 Series Genetic Analyzers
(8/24 Capillary)
3730 DNA Analyzer
(48/96 Capillary)

Data Analysis

SBTengine is a DNA-sequence analysis software package intended for high-resolution identification of alleles of the Human Leukocyte Antigens (HLA) generated through capillary sequencing. SBTengine® is intended for in vitro diagnostic use by professional health care personnel, such as laboratory technicians and physicians able to work according to EFI or ASHI specifications, or trained in HLA-typing and DNA sequencing in either EFI or ASHI accredited diagnostic laboratories.


NGS

HLA Typing by NGS (Next Generation Sequencing)

Over the past decade much has developed in the field of Next Generation Sequencing (NGS), starting from targeted sequencing to whole genome sequencing, the same has been adapted for studies of HLA genes.

  • which are complex in nature and cannot be comprehensively elucidated by traditional sanger sequencing.
  • To understand the HLA complexity, NGS facilitates complete HLA sequencing which will help researchers to have high-throughput and high-resolution data.

DNA Extraction

Our solutions for DNA extraction for HLA workflow are as follows:

JetQuick™ Blood and Cell Culture DNA Maxiprep Kit (Invitrogen™)
KingFisher™ Pure DNA Blood Kit (Thermo Scientific™)
DNAzol™ BD Reagent, for isolation of genomic DNA from whole blood (Invitrogen™)
GeneJET Whole Blood Genomic DNA Purification Mini Kit (Thermo Scientific™)
JetFlex™ Genomic DNA Purification Kit (Invitrogen™)
GeneCatcher™ gDNA Automated Blood Kit (Invitrogen™)
PureLink™ Genomic DNA Mini Kit (Invitrogen™)
iPrep™ PureLink™ gDNA Blood Kit (Invitrogen™)
To learn more about Nucleic acid isolation visit our Workflow

Target generation NGSgo®-IndX

NGSgo®-AmpX: HLA target generation

  • HLA locus-specific amplification primers for 12 loci
  • Single PCR per locus
  • Any combination of primers possible
  • Available as CE-IVD and RUO
  • NGS platform-independent
  • Crucial step of NGS workflow
  • Both alleles present in balanced manner

Library preparation NGSgo®-LibrX

  • Fragmentation, end repair and dA-tailing
  • Adapter ligation
    – X adapter contains barcode
    – P adapter contains ISP binding site
  • GenDx Adapters
    – X adapter T overhang
    – more specific – higher mappability/ Read depth

Clonal amplification Ion Chef

  • DNA fragments bind Ion Spear Particles (ISPs) with P adapter
  • Amplification of bound DNA fragments
  • Biotinylation of X adapter
  • Streptavidin coated magnetic bead bind to biotinylated DNA
Ion Chef
Chip loading

Sequencing Ion S5

The Ion Torrent semiconductor sequencing technology is based on the release of a hydrogen ion upon incorporation of a nucleotide into a strand of DNA by a polymerase. When a nucleotide is incorporated into a strand of DNA, a hydrogen ion will be released. The charge from that ion will change the pH of the solution, which can be detected by an ion sensor.

The sequencer sequentially floods the chip with one nucleotide after another in a repeated pattern of TACG. If a nucleotide that floods the chip is not incorporated, no pH change will be recorded and no base will be called. If there are two identical bases on the DNA strand, the pH change is twice as large, and the chip will record two identical bases.

Data Analysis NGSengine

  • NGS platform independent
  • One button analysis
    • Automatic HLA gene identification
    • Automated allele assignment
  • Alignment statistics

Post-Transplant Monitoring

When a human transplant is performed, antibody-mediated allograft injury caused by donor HLA-specific antibodies (DSA) is considered a major cause of late graft rejection. HLA molecules from a donor are recognized by the recipient’s immune system by direct and indirect methods of Allorecognition triggering an Alloimmune response. Hence, monitoring patients for DSA post-transplant and treating the same is an important step.

To help researchers and doctors with post-transplant monitoring we provide the following solutions:

Antibody ScreeningChimerism Monitoring

Antibody Screening

The micro lymphocyte toxicity test (MLCT) is a procedure which has been used for many years in HLA antibody diagnostics. Herein, anti-HLA antibodies from the patient’s serum (if available) react with the corresponding membrane bound antigens of the human lymphocytes which are fixed on the microtest plates.

Determining HLA antibodies is required in the preparation and progress monitoring of solid organ transplants. HLA class I antibodies can also influence the progression of thrombocyte transfusions. SeraScreen microtest plates contain ready to use pre-dripped peripheral lymphocytes from various donors to detect HLA antibodies. The plates are supplied with rabbit complement.

HLA antibody diagnostics: SeraScreen FCT microtest plates with deep frozen HLA-ABC typed lymphocytes

SeraScreen FCT microtest plates
Product Description Package REF PDF
SeraScreen FCT 60 (incl. rabbit complement) 5 plates à 1 Tests 7250 PDF
SeraScreen FCT 30 (incl. rabbit complement) 5 plates à 2 Tests 7251 PDF
HISTOPREP
Isolation of lymphocytes
Gen Trak Frozen Cell tray with pre-dropped anti-HLA seraRabbit complementRead under a fluorescence microscope

Chimerism Monitoring

In order to have a successful transplant and an appropriate therapy, monitoring for negative effects such as disease relapse, graft rejections and graft-versus-host disease is important. In this context, chimerism and mixed chimerism (MC) testing is the most suitable method in monitoring post-transplant outcome.

KMRtype® & KMRtrack® Chimerism Monitoring Reagents

  • Reduced lab workflow
  • Simultaneous typing of 8 Samples
  • Multiple donor analysis
  • Compatible with multiple real time thermo cyclers

KMRengine® Chimerism Analysis Software

  • Automated data analysis
  • Longitudinal data storage and reporting
  • Protocol generation and flexible experiment setup
  • Enables direct importing into LIMS

HLA-KMR™ HLA loss detection assay’s

  • Most frequent allele groups of HLA-A, C and DPB1
  • Applicable with KMRtrack protocol
  • Fully integrated with KMRengine

Chimerism monitoring is a two step decision process in the clinic:

Pretransplant Genotyping to identify Informative markers in prospective donors Posttransplant monitoring carried out monthly to evaluate which fraction of the transplant consists of recipient cells

1. Genotyping

2. Monitoring

We offer solutions for genotyping and monitoring using qPCR techniques

Software guidance – KMRengine

  • Compatible with all major qPCR machines
  • From protocol to data analysis
    • Supports experimental set-up
    • Creating protocols
    • Export and import files
    • Analyzes multiple donors
    • Typing and tracking experiments
    • Stores data

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