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Protocol - Human Leukocyte Antigen (HLA) Genotyping - Assay

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Description

This protocol includes instructions for drawing, processing and storing blood according to the Type 1 Diabetes Genetics Consortium (T1DGC) Manual of Operations. Human Leukocyte Antigen is measured by using the linear array genotyping system method.

Specific Instructions

The PhenX Infectious Diseases and Immunity Working Group would like to note that the science of HLA matching is rapidly advancing and evolving and multiple methods for HLA assays are available.

To aid in comparability, the Infectious Diseases and Immunity Working Group recommends that the investigator record the make and manufacturer of equipment used and the repeatability and coefficients of variation for the assay.

Please also note that the immobilized linear arrays used to genotype the samples were provided by Roche Molecular Systems and are not commercially available.

Availability

Available

Protocol

The protocol for blood collection and processing is found in the Type 1 Diabetes Genetics Consortium Blood Collection and Processing Manual of Operations (T1DGC Manual of Operations). The protocol for the extraction of DNA from whole blood is found in Rosinger et al. 2010. The protocol for human leukocyte antigen genotyping is found in Mychaleckyj et al 2010. Please note that the immobilized linear arrays used to genotype the samples were provided by Roche Molecular Systems and are not commercially available.

The following is a summary version of the full Type 1 Diabetes Genetics Consortium protocols. It is not intended to replace the actual protocols listed above.

Venipuncture / Blood Collection Procedures

Editors Note: Please review chapter VI of the Type 1 Diabetes Genetics Consortium Blood Collection and Processing Manual of Operations (T1DGC Manual of Operations - Chapter VI) for a full description of Phlebotomy procedures.

General steps:

  • Blood is collected from the best available vein.
  • Blood for DNA analysis should be collected in a 4.9-mL purple top tube (EDTA)
  • The tubes are handled in a way that prevents hemolysis
  • The tube contents are mixed by inverting eight times
  • Record date and time of blood collection
  • Record the reason why a blood sample was not collected.

Blood Processing

Editors Note: Please review chapter VI of the Type 1 Diabetes Genetics Consortium Blood Collection and Processing Manual of Operations (T1DGC Manual of Operations - Chapter VI) for a full description of Blood Processing procedures.

  • The sample is placed in an ice and water bath. Incubate the sample between 30 and 60 minutes.
  • Do not let any of the samples stand in direct sunlight or at extreme temperatures.
  • The sample is centrifuged, to separate the plasma from the cells.
  • The plasma is pipetted away from the sample without disturbing the cell pack.
  • The cell pack is shipped at ambient temperature to the DNA repository for DNA extraction.

DNA Extraction from Whole Blood

Please review Rosinger et al., 2010 for the full description of the DNA extraction protocol.

  • DNA is isolated by a modified salting out procedure or by cholorform extraction
  • DNA concentration is determined by fluorescence using a double-stranded DNA quantification reagent.
  • DNA quality is confirmed by comparison to an appropriate standard (ladder) on an agarose gel.

Laboratory Assay for Human Leukocyte Antigen

Please review Mychaleckyj et al., 2010 for a full description of the HLA genotyping methods. Please note that the immobilized linear arrays used to genotype the samples were provided by Roche Molecular Systems and are not commercially available.

  • DNA (5 ug total: 250 ul at 20ng/uL) is shipped to the HLA genotyping laboratory in screw top tubes.
  • Each human leukocyte antigen region is amplified from 60 ng of genomic DNA by polymerase chain reaction (PCR) on a separate 96 well plate according to standardized protocol (see Mychaleckyj et al., 2010 for details of the polymerase chain reaction reaction)
  • Polymerase chain reaction products are hybridized to oligonucleotide probes attached to nylon-backed membranes. The probes correspond to specific human leukocyte antigen DNA sequences. (Please note that the immobilized linear arrays used to genotype the samples were provided by Roche Molecular Systems and are not commercially available.)
  • Hybridized probes are visualized and assigned a genotype by software.
Personnel and Training Required

Phlebotomist

Laboratory capable of performing linear array genotyping

Equipment Needs

Phlebotomy supplies

Requirements
Requirement CategoryRequired
Major equipment Yes
Specialized training Yes
Specialized requirements for biospecimen collection No
Average time of greater than 15 minutes in an unaffected individual No
Mode of Administration

Bioassay

Lifestage

Toddler, Child, Adult

Participants

Children and Adults, 1 year and older.

Selection Rationale

The Type 1 Diabetes Genetic Consortium protocol was selected as the best standardized methodology for blood collection, processing and storage for the human leukocyte antigen genotyping assay. This protocol was used to genotype HLA-A, B, C, DRB1, DQ and DP loci in over 15,000 study participants between four different laboratory over five years.

Language

Chinese, English

Standards
StandardNameIDSource
Logical Observation Identifiers Names and Codes (LOINC) Assay human leukocyte antg (HLA) proto 62875-0 LOINC
Human Phenotype Ontology Abnormal leukocyte count HP:0001881 HPO
caDSR Form PhenX PX160601 - Assay For Human Leukocyte Antigen Hla Genotyping 6185122 caDSR Form
Derived Variables

None

Process and Review

Not applicable.

Protocol Name from Source

Type 1 Diabetes Genetics Consortium (T1DGC), Manual Of Operations. Blood Collection and Processing, 2010

Source

NIH. NIDDK. Type 1 Diabetes Genetics Consortium Manual Of Operations. Blood Collection and Processing.

Silke Rosinger, Sarah Nutland, Eric Mickelson, Michael D Varney, Bernard O Boehm, Gary J Olsem, John A Hansen, Ian Nicholson, Joan E Hilner, Letitia H Perdue, June J Pierce, Beena Akolkar, Concepcion Nierras, Michael W Steffes and T1DGC. Collection and processing of whole blood for transformation of peripheral blood mononuclear cells and extraction of DNA: the Type 1 Diabetes Genetics Consortium. Clinical Trials. 2010; 7: S65-S74.

Josyf C Mychaleckyj, Janelle A Noble, Priscilla V Moonsamy, Joyce A Carlson, Michael D Varner, Jeff Post, Wolfgang Helmberg, June J Pierce, Persia Bonella, Anna Lisa Fear, Eva Lavant, Anthony Louey, Sean Boyle, Julie A Lane, Paul Sali, Samuel Kim, Rebecca Rappner, Dustin T Williams, Letitia H Perdue, David M Reboussin, Brian D Tait, Beena Akolkar, Joan E Hilner, Michael W Steffes, Henry A Erlich and T1DGC. HLA genotyping in the international Type 1 Diabetes Genetics Consortium. Clinical Trials. 2010; 7: S75-S87.

General References

None

Protocol ID

160601

Variables
Export Variables
Variable Name Variable IDVariable DescriptiondbGaP Mapping
PX160601_Assay_Repeatability
PX160601090000 Repeatability of the assay N/A
PX160601_Blood_Draw_Comments
PX160601030200 Record any comments about the blood draw, more
including any reasons for the tube not being drawn according to the protocol. show less
Variable Mapping
PX160601_Blood_Draw_Done
PX160601030000 Was blood drawn? Variable Mapping
PX160601_Blood_Draw_Sample
PX160601030100 Was full amount obtained? N/A
PX160601_Coefficient_Of_Variation
PX160601100000 Coefficient of variation for the assay N/A
PX160601_Date_Of_Blood_Collection_Day
PX160601010200 Date of blood Collection - day N/A
PX160601_Date_Of_Blood_Collection_Month
PX160601010100 Date of blood Collection - month N/A
PX160601_Date_Of_Blood_Collection_Year
PX160601010300 Date of blood Collection - year N/A
PX160601_DNA_Concentration
PX160601050000 DNA concentration N/A
PX160601_DNA_Quality
PX160601060000 Enter comments to describe DNA quality N/A
PX160601_Equipment_Make
PX160601080100 Make of the equipment used to perform the more
HLA assay. show less
N/A
PX160601_Equipment_Manufacturer
PX160601080200 Manufacturer of the equipment used to more
perform the HLA assay. show less
N/A
PX160601_Genotyping_Results
PX160601110000 Results of the genotyping assay N/A
PX160601_HLA_Assay_Type
PX160601070000 Record the type of assay used for HLA testing. N/A
PX160601_Sample_Comments
PX160601040000 Record any comments about the sample during more
processing. show less
Variable Mapping
PX160601_Time_Of_Blood_Collection_AMPM
PX160601020300 Time of blood collection - am or pm N/A
PX160601_Time_Of_Blood_Collection_Hour
PX160601020100 Time of blood collection - hour N/A
PX160601_Time_Of_Blood_Collection_Minutes
PX160601020200 Time of blood collection - minutes N/A
Infectious Diseases and Immunity
Measure Name

Human Leukocyte Antigen (HLA) Genotyping Assay

Release Date

November 12, 2010

Definition

A bioassay to obtain the specific allelic genotypes of several of the human leukocyte antigens, whose genes reside on chromosome 6p within the Major Histocompatibility Region (MHC).

Purpose

The measure determines the alleles/single nucleotide polymorphisms that are present in the human leukocyte antigen regions of chromosome 6 to assess the major histocompatability system. The genetic variation within the human leukocyte antigen region determines the person's immune response and has been tied to specific autoimmune disorders such as Graves' disease and Hashimoto's thyroiditis, as well as other diseases such as multiple sclerosis, ankylosing spondylitis, and type 1 diabetes. This assay is similar to the genotyping performed in genome wide association studies but captures much more of the genetic variation within the human leukocyte antigen region than would be covered with "tag" single nucleotide polymorphisms.

Keywords

Infectious disease, Human leukocyte antigen, HLA, Type 1 Diabetes Genetics Consortium, Infectious Diseases and Immunity

Measure Protocols
Protocol ID Protocol Name
160601 Human Leukocyte Antigen (HLA) Genotyping - Assay
Publications

There are no publications listed for this protocol.