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).
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).
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)
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).
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
DNA Extraction and Quantification
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.
||HISTO TYPE A low
||HISTO TYPE B low
||HISTO TYPE C low
||HISTO TYPE DR low
||HISTO TYPE DQB low
||HISTO TYPE ABC
||HISTO TYPE DR/DQB
||HISTO TYPE ABDR
||HISTO TYPE DQB high
||HISTO TYPE B27 low
||HISTO TYPE B27 low
||HISTO TYPE B*57:01 / B*51
||HISTO TYPE Celiac Disease
||HISTO TYPE Narcolepsy
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.
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
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
|HISTO SPOT® A Xtend
|HISTO SPOT® B 4D
|HISTO SPOT® B Xtend
|HISTO SPOT® C 4D
|HISTO SPOT® DRB1 4D
|HISTO SPOT® DRB1 Xtend
|HISTO SPOT® DRB 3/4/5
|HISTO SPOT® DQB1 4D / DQA
|HISTO SPOT® DPB1
|HISTO SPOT® DPB1
|HISTO SPOT® Coeliac Disease
|HISTO SPOT® On-call Typing Kit
HISTO SPOT® Locus specific tests
|HISTO SPOT® Conjugate
|HISTO SPOT® Blocking Buffer
|HISTO SPOT® TBS Wash Buffer
|HISTO SPOT® Stringent Wash Buffer
|HISTO SPOT® Hybridization Buffer
|HISTO SPOT® Substrate
|HISTO SPOT® Mastermix A (on request)
|HISTO SPOT® Mastermix B (on request)
|HISTO SPOT® Mastermix C (on request)
|HISTO SPOT® Mastermix DRB1 (on request)
|HISTO SPOT® Mastermix DQ (on request)
|HISTO SPOT® Mastermix DPB1 (on request)
|HISTO SPOT® Mastermix DRB3/4/5 (on request)
|HISTO SPOT® Mastermix Coeliac Disease (on request)
|HISTO SPOT® Magnesium Chloride
HISTO SPOT® Hybridization reagents
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
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.
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
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:
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.
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.
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.
Target generation NGSgo®-IndX
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
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
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:
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
|SeraScreen FCT 60
||(incl. rabbit complement)
||5 plates à 1 Tests
|SeraScreen FCT 30
||(incl. rabbit complement)
||5 plates à 2 Tests
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
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