In the development of cancer immunotherapies, vaccines, and autoimmune disease treatments, researchers are increasingly aware of the importance of host human leukocyte antigens (HLA) in understanding the complexity of immune responses. HLA allelic variants have distinct binding affinities for peptide antigens during presentation, and thus regulate auto-antigen and pathogen-specific T cell activation. HLA polymorphisms have been correlated with disease risk, immune responses to infectious disease, as well as adverse responses to therapies. In this webinar, we will review the genetics and biology of HLA types and explain the technology underlying HLA identification. We will also provide some examples of how HLA-typing can be a valuable investment during early stage drug-development in human primary cells.
The angiogenesis process, the factors regulating it, different assays for it, a little about tumour angiogenesis, the drugs and new therapeutic approaches towards inhibiting or augmenting the process.
In the development of cancer immunotherapies, vaccines, and autoimmune disease treatments, researchers are increasingly aware of the importance of host human leukocyte antigens (HLA) in understanding the complexity of immune responses. HLA allelic variants have distinct binding affinities for peptide antigens during presentation, and thus regulate auto-antigen and pathogen-specific T cell activation. HLA polymorphisms have been correlated with disease risk, immune responses to infectious disease, as well as adverse responses to therapies. In this webinar, we will review the genetics and biology of HLA types and explain the technology underlying HLA identification. We will also provide some examples of how HLA-typing can be a valuable investment during early stage drug-development in human primary cells.
The angiogenesis process, the factors regulating it, different assays for it, a little about tumour angiogenesis, the drugs and new therapeutic approaches towards inhibiting or augmenting the process.
History
Introduction
Classification of grafts
The Immunology of Allogeneic Transplantation
Genetics of graft rejection
Types of rejection
Recognition of Alloantigens
Effector Mechanisms of Allograft Rejection
Prevention of graft rejection
Graft versus host reaction
Background of organ transplant infrastructure in the US. Some history. Definitions. Nursing Care of the transplant patient in hospital, and home settings. Intended for senior level nursing students in an ADN program
Case Study: Sequence-based HLA Typing - How I stopped worrying and started lo...Thermo Fisher Scientific
Wake Forest HLA Lab presents the issues and challenges that provoked thoughts of changing from SSP to SBT (Sequence-based typing), reveals lessons learned during implementation and shares their personal experience of working with Life Technologies during the installation of their 3500 XL Genetic Analyzer.
Visit the Life Technologies website to learn more about HLA Typing. http://owl.li/eedmi
How the Immune System Protects Your BodyPeter Corless
This is a presentation for laypersons or high schoolers who would want to understand the nature of pathogens, and how the human immune system works in response to such pathogens. It is not intended to be professional medical advice. Please seek further information from the U.S. Center for Disease Control (CDC), the World Health Organization (WHO), your local healthcare provider or a medical professional.
TRANSPLANTATION IMMUNOLOGY- MLR, HLA TYPING.pptxBharath S R
PURPOSE OF HLA TYPING, CONDITIONS THAT REQUIRING TRANSPLANTATION, THE PROCESS OF HLA TYPING, HLA TYPING IMPORTANT ROLE, SEROLOGICAL TEST, Microlymphocytotoxic test, MIXED LYMPHOCYTE REACTION, Molecular HLA typing, PCR BASED METHODS/ THREE CATEGORIES, STEPS OF MOLECULAR CLONING, Sequence specific priming, Hybridization with sequence specific oligonucleotide probes (SSOP), SEQUENCE BASED HLA TYPING, CLINICAL SIGNIFICANCE OF HLA TYPING,
Question 1 200-300 words..cite sourcesChoose one of the five im.docxIRESH3
Question 1: 200-300 words..cite sources
Choose one of the five immunoglobulin isotypes and discuss biological properties related to your selected isotype. What clinical effects would you expect if your body no longer produced this isotype? How could these effects be mediated?
Paper
Read the “Tools for Human Leukocyte Antigen Antibody Detection and Their Application to Transplanting Sensitized Patients” article in this week’s Electronic Reserve Readings.
Write a 450 to 700-word paper that includes the following:
Analyze the current challenges with organ transplantation related to the genetic variability of MHC.
Describe the challenges of human leukocyte antigen (HLA) matching and identifying sensitized individuals.
Format your paper consistent with APA guidelines. Use at least 3 to 5 different references in your paper.
Transplantation
Issue: Volume 86(3), 15 August 2008, pp 384-390
Copyright: (C) 2008 Lippincott Williams & Wilkins, Inc.
Publication Type: [Editorials and Perspectives: Overview]
DOI: 10.1097/TP.0b013e31817c90f5
ISSN: 0041-1337
Accession: 00007890-200808150-00003
Keywords: Renal transplantation, HLA antibody detection, Sensitized
patients
[Editorials and Perspectives: Overview]
Tools for Human Leukocyte Antigen Antibody Detection and Their Application to
Transplanting Sensitized Patients
Fuggle, Susan V.1,2,3,5; Martin, Susan4
Author Information
1 Transplant Immunology and Immunogenetics, Oxford Transplant Centre, Churchill
Hospital, Oxford, United Kingdom.
2 Nuffield Department of Surgery, University of Oxford, Oxford, United Kingdom.
3 UK Transplant, Bristol, United Kingdom.
4 Transplantation Laboratory, Manchester Royal Infirmary, Manchester, United
Kingdom.
5 Address correspondence to: Susan V. Fuggle, Ph.D., Transplant Immunology and
Immunogenetics, Oxford Transplant Centre, Churchill Hospital, Oxford OX3 7LJ,
United Kingdom.
E-mail: [email protected]
Received 8 January 2008. Revision requested 11 February 2008.
Accepted 14 April 2008.
----------------------------------------------
Outline
Abstract
Antibody Identification
Complement Dependent Cytotoxicity
Flow Cytometry
Solid Phase Assays
Enzyme-Linked Immunosorbent Assay
Flow Cytometry
Luminex
Patient Sensitization Profile
Transplanting Sensitized Patients
ACKNOWLEDGMENTS
REFERENCES
Abstract
In recent years there have been major advances in the technology for the
detection and definition of human leukocyte antigen antibodies. In this overview
we describe the evolution in laboratory technology, the techniques currently
available and consider their application in antibody specificity definition and
in understanding a patient's sensitization profile. We discuss the importance of
antibody specificity definition in facilitating efficient national organ
allocation and informi ...
Towards Precision Medicine: Tute Genomics, a cloud-based application for anal...Reid Robison
Tute Genomics is cloud-based software that can rapidly analyze entire human genomes. The cost of whole genome sequencing is dropping rapidly and we are in the middle of a genomic revolution. Tute is opening a new door for personalized medicine by helping researchers & healthcare organizations analyze human genomes.
What is in situ hybridization
Radioactive ISH
Fluorescent ISH
Colorimetric ISH
ISH: three variables
The sample
The probe
Optimizing ISH Detection
ISH controls
Data Analysis
Summary table that compares the performance specifications of Illumina's new sequencers - HiSeq X and NextSeq with MiSeq and HiSeq 2500. Includes number of samples that can run on the different sequencers.
New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
Associate Division Director for Ambulatory Operations
The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
HOT NEW PRODUCT! BIG SALES FAST SHIPPING NOW FROM CHINA!! EU KU DB BK substit...GL Anaacs
Contact us if you are interested:
Email / Skype : kefaya1771@gmail.com
Threema: PXHY5PDH
New BATCH Ku !!! MUCH IN DEMAND FAST SALE EVERY BATCH HAPPY GOOD EFFECT BIG BATCH !
Contact me on Threema or skype to start big business!!
Hot-sale products:
NEW HOT EUTYLONE WHITE CRYSTAL!!
5cl-adba precursor (semi finished )
5cl-adba raw materials
ADBB precursor (semi finished )
ADBB raw materials
APVP powder
5fadb/4f-adb
Jwh018 / Jwh210
Eutylone crystal
Protonitazene (hydrochloride) CAS: 119276-01-6
Flubrotizolam CAS: 57801-95-3
Metonitazene CAS: 14680-51-4
Payment terms: Western Union,MoneyGram,Bitcoin or USDT.
Deliver Time: Usually 7-15days
Shipping method: FedEx, TNT, DHL,UPS etc.Our deliveries are 100% safe, fast, reliable and discreet.
Samples will be sent for your evaluation!If you are interested in, please contact me, let's talk details.
We specializes in exporting high quality Research chemical, medical intermediate, Pharmaceutical chemicals and so on. Products are exported to USA, Canada, France, Korea, Japan,Russia, Southeast Asia and other countries.
Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
3. What is HLA?
• HLA = Human Leukocyte Antigen
• Immune cells read HLA-barcode on cells to help identify
self vs. non self cells or uninfected vs. infected cells.
– An immune response is triggered if a infected cell is identified.
Y
Y
Y
YY
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Infected cell
T-cell
Uninfected cell
Uninfected cell
Uninfected cell
4. • Any cell displaying some other HLA type is "non-self" and
is seen as an invader by the body's immune system,
resulting in the rejection of the tissue bearing those cells.
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Non-self cell : INVADER
T-cell
Self cell
Self cell
Self cell
What is HLA?
5. MHC Region
MHC Class II genes MHC Class III genes MHC Class I genes
HLA-AHLA-C
TNF C4
HLA-DQHLA-DP HLA-DR
HLA-B
MHC houses HLA genes
Chromosome 6
6. MHC Region
MHC Class II genes MHC Class III genes MHC Class I genes
HLA disease associations
Chromosome 6
- Leprosy
- Multiple Sclerosis
- Lymphoid Leukemia
- Rh(D) isoimmunization
- Psoriasis
- Ankylosing spondylitis
- Hemophilia with synovitis
- Malaria
- Susceptibility or Resistance to HIV-1
- Type1 autoimmune hepatitis
- ANCA-positive autoimmune disease
- Multiple Sclerosis
- Psoriasis
- Systemic Lupus
- Asthma
- Childhood Acute Lymphoblastic
Leukemia (ALL)
- HIV-related disease
- Thyroid Carcinoma
- Nephropathy
- Kawasaki disease
- Celiac Disease
http://ccg.murdoch.edu.au/private/yurek_kulski/mhc/Table4.html
8. HLA status and control over HIV infection
- HIV controllers = HIV positive individuals that do not
develop a clinical disease and do not progress to AIDS.
- HLA Class I SNPs can determine whether you are a HIV
controller or progressor http://aids.gov/hiv-aids-basics/hiv-aids-101/what-is-hiv-aids/
X
X
9. HLA drug hypersensitivity testing
Allergy to Abacavir is strongly associated with the
presence of the HLA-B*5701 gene
10. Gluten intolerance
• Celiac disease is an autoimmune disorder of
the small intestine triggered by gluten
consumption.
• Over 95% of people with celiac have the
isoform of DQ2 or DQ8, on Class II MHC
genes.
11. • Organ transplantation
• Hematopoietic stem cell transplant
• HLA types associated with specific immune
diseases.
• Parental testing
• Reduce instance of GVHD
– Non-related donor
– Related donor
Importance of HLA typing
12. Did you know?
• In the United States:
– Nearly 120,000 need lifesaving organ
transplants.
– Every 10 minutes another name is added to
the national organ transplant waiting list.
– An average of 6000 people die each year
from the lack of available organs for
transplant.
– In 2012, there were 14,013 Organ Donors
resulting in 28,052 organ transplants.
– More than 1 million tissue transplants are
done each year and the surgical need for
tissue has been steadily rising.
• $230M worldwide transplant test market.
13. 1, 8, 10
3, 14, 17
10, 16, 8
2, 7, 11
1, 8, 10 1, 8, 10
2, 7, 11 10, 16, 8
3, 14, 17
2, 7, 11
3, 14, 17
10, 16, 8
Mother Father
Daughter 1 Daughter 2Son 1 Son 2
10, 16, 8
Son 3
1, 8, 10
Variation in HLA alleles even between siblings.
Random and varied distribution of Haplotypes from each parent.
High resolution typing provides a more accurate match
Finding a match is not easy!
Match
14.
15. • Serotyping
– Detection of antibodies is serum (non
sequencing based method).
• Sanger sequencing of amplified regions
– Amplicon sequencing of specific exon regions.
• Sequence Specific Oligonucleotide
Hybridization (SSO)
– Oligo hybridization based detection of allele
status.
• (NGS) MHC sequencing for donor matching
– Sequencing of entire MHC region.
– Sequencing of amplicons for target regions.
Typing Methods
16. Method About method Pros Cons
Serotyping
Non-sequencing based typing method
where antibodies specific to HLA
proteins are used to identify the
proteins on the cell surface.
- Low Cost
- Rapid
- Traditional
- Crude Method
- Protein based detection
- Inaccurate typing
- Protein binding to more
than one serotype
Sequence Specific
Oligonucleotide
Hybridization (SSO)
Typing method where specific oligos are
first designed for genes of interest and
then hybridized to patient or donor
DNA to check for hybridization.
- Checking of specific
target
- Efficient
- Cannot account for
unrecorded alleles
- Hybridization errors
- Need to know target
sequence
- Cannot phase
Sanger Sequencing
Sanger sequencing or Sequencing by
Termination (SBT) is a classical method
used for sequencing specific regions of
the MHC.
- Used to sequence
regions of interest
- Fast
- Base pair resolution
- Coverage only 2x
- Different HLA alleles
share similar sequences,
difficulty aligning.
- Cannot phase
Next-Gen Seq
Performing long range PCR to amplify
HLA genes in MHC region, fragmenting
the amplified genes and then
preforming deep sequencing.
- Deep coverage (1000x)
- Total MHC coverage
- Rapid high throughput
- Accurate and efficient
- Phasing
- Data Analysis
Typing Methods
20. Table S1: PCR primers and estimated amplicon size for the 6 HLA genes
Locus Forward primer Seq Reverse primer Seq
Length
(bp)
HLA-A HLA-A_F7 ATCCTGGATACTCACGACGCGGAC HLA-A_R8 CATCAACCTCTCATGGCAAGAATTT 3398
HLA-C HLA-C_F4 GGCCGCCTGTACTTTTCTCAGCAG HLA-C_R3 CCATGGTGAGTTTCCCTGTACAAGAG 4440
HLA-B HLA-B_F3 AGGTGAATGGCTCTGAAAATTTGTCTC HLA-B_R3 AGAGTTTAATTGTAATGCTGTTTTGACACA 4296
HLA-DRB1 HLA-DRB1_F1 TGATTGACTTGCTGGCTGGTTTCTCATC HLA-DRB1_R2 GCATCCACAGAATCACATTTTCTAGTGTT 11899
HLA-DQB1 HLA-DQB1_F6 TCATGTGCTTCTCTTGAGCAGTCTGA HLA-DQB1_R7 TGTGACAGCAATTTTCTCTCCCCT 7118
HLA-DPB1 HLA-DPB1_F1 TGGTCCAACAGGATCACATTTATAAGTGT HLA-DPB1_R1 CCCAGTTTGGATGGTCTCTCAGCTCTT 13605
13,605bp 7,118bp 11,899bp
4,296bp 4,440bp 3,398bp
Long range PCR
Nextera
MiSeq
21. • Nextera/MiSeq
– Coverage of coding and non-coding HLA
regions.
– Nextera tagmentation protocol provides easier
faster processing of samples
– Greater depth delivers accuracy exceeding
existing technologies.
Sequencing
22. 1, 8, 10
3, 14, 17
10, 16, 8
2, 7, 11
1, 8, 10 1, 8, 10
2, 7, 11 10, 16, 8
3, 14, 17
2, 7, 11
3, 14, 17
10, 16, 8
Mother Father
Daughter 1 Daughter 2Son 1 Son 2
10, 16, 8
Son 3
1, 8, 10
Variation in HLA alleles even between siblings.
Random and varied distribution of Haplotypes from each parent.
Finding a match is not an easy task.
Unique HLA fingerprint
Match
23. • MiSeq install at HistoGenetics and DKMS Life Science Lab in
Germany.
High resolution HLA typing because:
– Read length (2x250, 2x300) allows phasing
– Highest accuracy and speed
– 1000x Coverage
• (consensus accuracy 99.999%)
– No Homo polymer errors
– Easier workflow
MiSeq leads the way
24. • Analyze SNPs, allelic differences,
status of microsatellites, and
HLA typing all in one.
• Basic SNP or genotyping
analysis tool
• IMGT-HLA database
Data Analysis
27. • Currently at about 50 HLA related MiSeq installs in the last few months
– DKMS. From Martin Allgaier’s install report: “DKMS Life Science Lab, a subsidiary of
DKMS, is a state-of-the-art HLA tissue typing laboratory in Dresden, Germany
performing all sequencing activities to MiSeq. This was the 11th (!!!) Miseq for this
customer and they are still planning for more. Currently, there are no signs of switching
to another platform as the MiSeq gives them the output and flexibility they need for
their service.”
– HistoGenetics. From Peter Kraus’ install report: “MiSeq Install Report for the first 10 of
20 total MiSeqs to be installed at HistoGenetics in Ossining, NY. HistoGenetics is a
company that performs HLA typing as a service using sequencing assays”
• Press release."We anticipate building on our leadership position and established
success by leveraging the MiSeq platform," said Dr. Nezih Cereb, Chief Executive
Officer and Co-founder of HistoGenetics. "Based on our experience with other
technologies, we think the MiSeq's quality data output and simple workflow,
combined with Illumina's commitment to work collaboratively, is the ideal solution
to enable us to provide our customers with the superior results and turnaround
time they require."
Example: MiSeq installs in HLA labs
28. • Research customers
– Association of HLA variants
with disease
• Inflammatory, infection
susceptibility, cancer, etc.
• More accurate cataloging of
HLA types
• Translational customers
• Tissue registries
• Marker validation for
diseases
• Clinical customers
• Transplantation match
– Registry
– Confirmatory
HLA sequencing: Business Opportunity
$230M worldwide
Software companies
Conexio • SW market leader. Sanger legacy
Omixon • FDA-compliance
GenDx • mature SW, nice UI
Editor's Notes
Hello everyone. Thank you for joining in. Today’s talk is titled HLA typing: Understanding the barcode on your cells. My goal is to introduce HLA, it’s importance, current methods, and how Illumina’s technology can offer a benefit over existing methods and technologies. So let’s begin…Our bodies are equipped with an amazing barcoding mechanism
Just like in our day to day lives we use barcodes to identify objects and define them, allowing easier recognition and organization.Our body uses a similar tool to help scan and organize our body such that objects that don’t belong there are tagged for immediate removal or destruction.
So what is HLA? HLA stands for Human Leukocyte Antigen.HLA acts like a barcode on each of our cells to help the scanners of our body, which are our immune cells identify barcodes from infected cells that do not belong in our body.
What is interesting is that such a mechanism not only helps identify infected cells, but it also sees “non-self” cells as invaders. What does that mean?… Any cell that displays another type of HLA that is not something your own DNA encodes for is targeted. This can happen when an organ from a donor who’s HLA type does not match your own is transplanted, leading to organ rejection or Graft versus host diseases.Now that we know what HLA is, lets take a closer look at the genes that encode these barcodes.
The Major Histocompatibility Complex (MHC) on Chromosome 6 houses Human Leukocyte Antigen (HLA) genes.The MHC region is distributed into three classes. Class I, Class II and Class III genes. Of which the Class II and Class I genes are important during transplantation to reduce the chances of rejection or disease. This MHC region is the most polymorphic region in the human genome, meaning there are multiple variants of each gene within the population as a whole.
What is interesting is that polymorphisms in this region associate with multiple diseases. I have listed a few here, most of which are autoimmune.
Here is a more comprehensive list of the diseases that associate with SNPs in specific regions of the MHC.I mean just look at the amount of information that is hidden within this region. A 4Mbp region can provide information and predict more than 70 disease states (a few examples are).-Height, Hypothyroidism, Bipolar disorder, Menopause, Lung cancer, -Arthiritis, Parkinsons, Total cholesterol-Type1 Diabetes, Nephropathy, Celiac disease-Kawesaki diseaseThe list goes on. It’s unimaginable how such a tiny region in the genome can show associations with so many diseases and disorders.
Polymorphisms in HLA have control over your susceptibility or protection from the HIV virus. The HIV virus attacks your T-cells (important part of your scanning system) and makes copies of it self. So the virus hijacks your scanner, which then compromises its ability to read barcodes.Research by the International HIV controllers Study performed a Genome-wide association analysis in multiethnic cohort of HIV-1 controllers and progressors and analyzed the effects of individual amino acids within the classical HLA proteins. Where more than 300 SNPs were located within the MHC and none else. So what are HIV controllers and progressors?HIV controllers are HIV positive patients that are less likely to transmit HIV to others and do not develop a clinical disease. They also maintain stable CD4+ cell counts (usually declines in HIV positive individuals as the disease progresses to AIDS). These 'HIV controllers' do not require treatment, because their bodies suppress the replication of the virus, since they do not develop AIDS.Polymorphisms within this region on the MHC can determine whether you are a controller (do not develop AIDS) or a progressor after a HIV infection.
Allergies to various drugs is also associated with specific SNPs in the MHC region.For those that are diagnosed as HIV positive are treated with a drug called Abacavir, but about 5-8% of HIV positive patients have hypersensitivity to anti-retroviral drug Abacavir, which is the first line of defense in HIV treatment.Currently a skin test that takes 24-48 hours is used to test for allergies and is not consistent between ethnic backgrounds. The test had a 44% sensitivity amongst white patients and 14% sensitivity amongst black patients with a clinically suspected hypersensitivity reaction. Sequencing on the other hand can provide more robust and clear indications of allergies to drugs.
Another disease that is associated with SNPs in the MHC is Celiac disease, which is an immune reaction to eating gluten, a protein found in wheat, barley and rye. Eating gluten triggers an immune response and damages the lining of the intestine. There's no cure for celiac disease — but following a strict gluten-free diet can help manage symptoms and promote intestinal healing. LabCorp provides sequencing services to sequence the HLADQ genes to determine celiac disease.
So far we have explored the role of HLA in disease associations, but wait there is more!HLA typing plays a crucial role during organ or stem cell transplant. It can also be used for Parental testing and to reduce the instance of Graft versus Host Disease (GVHD)
A Higher HLA typing resolution can assure fewer chances of organ rejection and more accurate patient-donor matching. This is pretty big with a 230M worldwide transplant test market.
Everyone’s HLA type is very unique. For instance a mother and father have a specific HLA type, their children then have the probability to have a distinct HLA type depending on which Haplotype they inherit, as you can see here it makes it even difficult for siblings to be a close match. High resolution HLA typing can provide a more accurate match in a shorter time frame, where with sequencing a larger database can be scanned for potential matches faster.
A novel by Jodi Picoult, My sister’s keeper, now a feature film, is about a small girl who was born as a donor for her older sister that was affected by a fatal disease. The story is about a savior sibling. Surprisingly, this is not a matter of fiction, but is real and is an option desperate parents make to save their children. Let’s talk about how a savior sibling is created. Multiple embryos are created and preimplantation genetic diagnosis is used to detect and select ones that are free of a genetic disorder and that are also a HLA match for an existing sibling who requires a transplant.
Serotyping is a non-sequencing based method, which detect the presence of certain antibodies in serum, providing information about HLA type.Sanger sequencing of specific exon regions can be performed for HLA typing informationA method called SSO or sequence specific oligonucleotide hybridization detects polymorphisms. DNA probes for various regions are spotted onto a membrane. Upon hybridization of designed oligos to patient DNA a detectable signal is given off, indicating a specific HLA genotype.For NGS methods either the entire MHC region can be sequenced or targettedamplicon sequencing can be performed, depending on what is being tested.
As you can see the nomenclature designations can be rather complex, reminding us how polymorphic and variable these genes can be.Each number is a digit and current methods are able to give information of upto 4 digits only.
More and more alleles are being identified as higher resolution technology becomes available. As we identify more alleles we can better understand their associations with diseases as well as better be able to identify a specific HLA type.This is pretty exciting, getting an indepth look on the HLA alleles provides a more comprehensive map of the MHC region.We have just scratched the surface and we a long ways away from identifying all the alleles present within the human population.
This is a great paper by Hosomichi et al provides a protocol for phase defined sequencing of HLA using Nextera and Illumina sequencing technology.
They performed long range PCR of regions within the MHC (highlighted in red) and used nextera kit to perform tagmentation and miseq for sequencing. Here we have primer information for those interested in using this protocol for analyses.This paper provides an easy and fast protocol that addresses multiple things:Provides high resolution HLA typing – which allowed the group to detect new allelesWe can get phasing informationInstead of the only 4-digit HLA information, we can now get resolution for upto 8-digits (almost double the information) – which means more accurate typing.
Just to conclude the Nextera/MiSeq approach.
Lets recall how difficult it is to find a match, where even finding a match within siblings can be extremely difficult. Faster processing of samples and greater more accurate coverage can allow for searching a larger repository of donors to find a close match.A mismatch can lead to diseases and or organ rejection. With such a shortage of organ donors and an ever increasing demand for organs, it becomes crucial to get accurate, higher resolution HLA typing information. Such that a correct match can be made, preventing further disease or organ rejection.
We have recently performed two large installs of the MiSeq. At HistoGenetics and DKMS Life science lab. HistoGenetics performshigh-resolution HLA Sequence-Based Typing (SBT) services. DKMS a subsidiary of DKMS life science lab is a tissue typing laboratory. So we can see that the demand and interest for high resolution of HLA typing is increasing.MiSeq offers a benefit over other technologies as it provides higher resolution:
Some ways to analyze the data is by simply performing basic SNP analysis using the IMGT-HLA database. I have some examples provided here, such as HLAminer and Omixion analysis tools, but there are many mature tools available which takes raw data and generates information about the HLA barcode in a semi-automated manner.
Just imagine the plethora of information we can get from deciphering the HLA-barcode. Current technology has now made it possible to dig deeper. We can predict diseases, improve organ transplant rates, and eventually save lives.So let’s crack the code with current tools and knowledge and decipher the HLA-barcode so we can better understand our body and what exactly our MHC has in store for us.