Transplant Biology & HLA

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)

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

PCR amplification

Gel Electrophoresis

Data Analysis

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

MR.SPOT Processor

DNA Extraction

Locus specific PCR amplification

Hybridization of amplicons to probes

Data Analysis

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

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:

Veriti™ 96-Well Fast Thermal Cycler

MicroAmp™ Fast Optical Plate

MicroAmp™ Optical Adhesive Film

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.

ExoSAP-IT™ PCR Product Cleanup Reagent

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

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.

NGSgo^®-AmpX

DNA Extraction

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

Data Analysis NGSengine

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

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

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.

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